Method for differentiating mesenchymal stem cells into neural stem cells and application

Abstract

The invention relates to a method for differentiating mesenchymal stem cells into neural stem cells and application, and belongs to the technical field of stem cell engineering.The method comprises the steps that (a) the mesenchymal stem cells are cultured in a culture medium containing an HDAC inhibitor and a DNA methyltransferase inhibitor to induce differentiation of the mesenchymal stem cells, and therefore cells for promoting expression of neural stem cell marker genes are formed; (b) culturing the cells obtained in step (a) in a culture medium containing an HDAC inhibitor, a DNA methyltransferase inhibitor, a retinoic acid receptor agonist and a GSK-3 inhibitor to induce differentiation thereof, thereby forming cells that initiate expression of a neural stem cell marker protein; and (c) culturing the cells obtained in step (b) in a culture medium containing a GSK-3 inhibitor and a retinoic acid receptor agonist to induce differentiation thereof, thereby forming neural stem cells. The differentiation method disclosed by the invention has the characteristics of short preparation period and high differentiation efficiency, and the prepared neural stem cells can be used for treating / or preventing neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of stem cell engineering technology, specifically relating to a method and application for differentiating mesenchymal stem cells into neural stem cells. Background Technology

[0002] Nerve injury refers to the disruption of the integrity of neural tissue leading to functional impairment. It is often caused by factors such as trauma, disease, tumors, genetics, or infection. Traditional treatments include surgery, physical therapy, and medication. With a deeper understanding of nerve injury, exploring nerve regeneration (i.e., tissue regeneration and functional recovery after nervous system injury through self-regeneration or external intervention) and nerve repair (using stem cells, biomaterials, gene therapy, and other technologies to promote repair) has become a medical hotspot. Stem cells, especially neural stem cells (NSCs) with self-renewal and multi-lineage differentiation potential, hold a crucial position and have broad prospects in the biomedical field. As pluripotent cells, NSCs can develop into neural lineage cells. After transplantation to the site of spinal cord injury (SCI), they can differentiate into nerve cells to replace damaged cells, provide nutritional support, rebuild connections, and promote regeneration, making them an important strategy for SCI repair. More broadly, neural stem cells have the ability to differentiate into neurons, glial cells, etc., providing a highly promising new approach for treating neurological diseases such as Parkinson's disease, Alzheimer's disease, and stroke.

[0003] In recent years, research on stem cell-to-neural stem cell therapy has made significant progress, with multiple clinical cases and animal experiments providing new treatment strategies for neurodegenerative diseases and injuries. In 2024, a team at Peking Union Medical College Hospital completed the world's first Phase I clinical trial of neural stem cell transplantation via the nasal mucosa for the treatment of Parkinson's disease. This study enrolled 18 patients with intermediate to advanced stages of the disease, delivering human neural stem cells to the brain through the olfactory nerve pathway in the nasal cavity, avoiding the risks of traditional invasive surgery. A 12-month follow-up showed significant improvement in patients' motor function scores (MDS-UPDRS), particularly a 19.9-point decrease in the average score at 6 months post-treatment, with no serious adverse reactions observed. [1] In 2023, *Nature Medicine* reported a phase I clinical trial for progressive multiple sclerosis (PMS). The study, involving intrathecal injection of neural stem cells, slowed the progression of the Extended Disability Status Scale (EDSS) score and significantly reduced the rate of brain atrophy in 12 patients. Two-year follow-up showed that the highest-dose group had significantly better percentage change in brain volume (PBVC) than the control group, and no serious adverse events occurred. This study provides important evidence for the application of neural stem cells in demyelinating diseases. [2] .

[0004] Currently, the induction of mesenchymal stem cells (MSCs) into neural stem cells (NSCs) mostly uses growth factor induction (such as bFGF / EGF), which has problems such as low efficiency (reported in the literature to be about 30 - 40%) and long cycle (14 - 21 days). There are defects such as incomplete differentiation and low cell survival rate in the direct chemical induction method of mesenchymal stem cells (MSCs) into neural stem cells (NSCs). [3] .

[0005] As an important member of the adult stem cell family, adipose-derived mesenchymal stem cells (ADMSCs) are rich in sources, easy to obtain, have low trauma, strong in vitro proliferation ability, and low culture cost. In addition, ADMSCs have no risk of tumorigenicity. Different from the risk of teratoma formation in embryonic stem cells or gene mutation risk in iPSCs, the safety of ADMSCs has been verified in more than 2000 clinical studies.

[0006] Therefore, there is an urgent need in this field to develop a method for efficiently inducing adipose-derived mesenchymal stem cells (ADMSCs) to differentiate into neural stem cells (NSCs) through a combination of small molecule compounds in stages.

[0007] References: JIANG S, WANG H, YANG C, et al. Phase 1 study of safety and preliminary efficacy of intranasal transplantation of human neural stem cells (ANGE-S003) in Parkinson's disease[J]. J Neurol Neurosurg Psychiatry, 2024, 95(12): 1102 - 11. GENCHI A, BRAMBILLAE, SANGALLI F, et al. Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study[J]. Nat Med, 2023, 29(1): 75 - 85. CHENG Feng, HAO Huaiyong, TIAN Heping, et al. Study on the differentiation and apoptosis of bone marrow mesenchymal stem cells into neural cells in vitro[J]. Acta Universitatis Medicinalis Nanjing (Natural Science Edition), 2010, (01): 54 - 8. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for differentiating adipose-derived mesenchymal stem cells into neural stem cells using the patient's own adipose-derived mesenchymal stem cells. This preparation method can greatly reduce the risk of recipient rejection and increase the success rate of surgery. Furthermore, this differentiation method has the characteristics of short preparation cycle, high differentiation efficiency, and improved safety performance.

[0009] In a first aspect, the present invention provides an in vitro culture method for generating neural stem cells from mesenchymal stem cells, the method comprising the following steps: (a) Mesenchymal stem cells were cultured in a medium containing HDAC inhibitors and DNA methyltransferase inhibitors to induce their differentiation, thereby forming cells that initiate the expression of neural stem cell marker genes; (b) The cells obtained in step (a) were cultured in a medium containing an HDAC inhibitor, a DNA methyltransferase inhibitor, a retinoic acid receptor agonist and a GSK-3 inhibitor to induce their differentiation, thereby forming cells that initiate the expression of neural stem cell marker proteins. (c) The cells obtained in step (b) are cultured in a medium containing a GSK-3 inhibitor and a retinoic acid receptor agonist to induce their differentiation, thereby forming neural stem cells.

[0010] Optionally, the concentration of the HDAC inhibitor in step (b) is lower than the concentration of the HDAC inhibitor in step (a); the concentration of the DNA methyltransferase inhibitor in step (b) is lower than the concentration of the DNA methyltransferase inhibitor in step (a); and step (c) does not contain either the HDAC inhibitor or the DNA methyltransferase inhibitor.

[0011] Optionally, the concentration of the HDAC inhibitor and / or DNA methyltransferase inhibitor described in step (b) is at least 2, 5, or 10 times lower than that in step (a).

[0012] Optionally, the HDAC inhibitor is one or more of trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entenstatin (MS-275).

[0013] Optionally, the DNA methyltransferase inhibitor is one or more of 5-azacytidine (5-AZA), RG108, Decitabine, and SGI-1027.

[0014] Optionally, the retinoic acid receptor agonist is one or more of retinoic acid, AM580, all-trans retinoic acid (ATRA), TTNPB, and AC55649.

[0015] Optionally, the GSK-3 inhibitor is one or more of CHIR99021, BIP-135, CP21R7, 1-Azakenpaullone, TDZD-8, SB216763, SB415286, BIO-acetoxime, Tideglusib, and AR-A014418.

[0016] Optionally, the HDAC inhibitor is TSA, the DNA methyltransferase inhibitor is 5-AZA, the retinoic acid receptor agonist is ATRA, and the GSK-3 inhibitor is CHIR99021.

[0017] Optionally, the method further includes the step of: (d) The neural stem cells obtained in step (c) are cultured in a neural basal medium to further mature and proliferate the neural stem cells.

[0018] Optionally, the culture medium in steps (a)-(c) may further comprise a neural basal culture medium.

[0019] Optionally, the neural basal culture medium consists of DMEM / F12, B27, N2, ITS, ascorbic acid, EGF, and bFGF.

[0020] Optionally, the mesenchymal stem cells are adipose-derived mesenchymal stem cells (ADMSCs).

[0021] In a second aspect, the present invention provides a neural stem cell or a population thereof generated by or directly from the method described in the first aspect.

[0022] Thirdly, the present invention provides a composition comprising neural stem cells or a population thereof as described in the second aspect.

[0023] Fourthly, the present invention provides the use of neural stem cells or cell populations thereof (as described in the second aspect) or compositions (as described in the third aspect) in the preparation of medicaments for the treatment and / or prevention of neurodegenerative diseases.

[0024] Fifthly, the present invention provides a kit for in vitro differentiation and culture of neural stem cells, comprising (a) at least one HDAC inhibitor, (b) at least one DNA methyltransferase inhibitor, (c) a retinoic acid receptor agonist, and (d) a GSK-3 inhibitor, optionally, the kit further comprising a neural basal culture medium.

[0025] This invention includes at least one of the following beneficial technical effects: 1. By using pulsed stage induction, the epigenetic barrier of direct transdifferentiation of MSCs is avoided, making the process more reasonable; 2. Compared with traditional single neurotrophic factor induction, it significantly improves the transformation rate of neural stem cells, while increasing cell safety and ensuring cell viability; 3. In the first stage, gene silencing is lifted, and in the second stage, the effect is maintained. At the same time, the expression of characteristic genes is activated to maximize the efficacy of small molecule compounds. Attached Figure Description

[0026] Figure 1 The morphological changes of neural stem cells in stage four; Figure 2 Immunohistochemical images show PAX6 (A), Nestine (B), β-III-Tubulin (C), and SOX2 (D), respectively. Figure 3 This is an immunofluorescence staining image of β3-tubulin in the NSCs of this application; Figure 4 This is a Nestine immunofluorescence staining image of the NSCs in this application. Detailed Implementation

[0027] The term "adipose-derived mesenchymal stem cells" (also known as ASCs, ADSCs, ADMSCs) refers to a type of somatic stem cell found in adipose tissue that has the ability to self-renew and differentiate into various cell types.

[0028] In this application, the basal induction culture medium consists of DMEM / F-12, B27, ITS, N2, ascorbic acid, bFGF, and EGF. bFGF and EGF are neurotrophic factors that can serve as basal culture components, activating the MAPK / ERK pathway, promoting neural progenitor cell proliferation, upregulating Nestin transcription, maintaining the proliferative capacity of neural stem cells, and synergistically inducing neural progenitor cell maturation.

[0029] This application pioneers a "pulse-based, multi-stage" differentiation-inducing method: a) First stage: Adding DNA methyltransferase inhibitors and histone deacetylase inhibitors can unleash the silencing of promoter genes such as PAX6, Nestine, Tubbs, and SOX2, promoting the opening of related genes; In one specific embodiment of the first stage, the culture medium comprises neural basal medium as a basal medium and further comprises 5-AZA and TSA. In one embodiment, the culture medium contains about 100 nM to about 300 nM of TSA, preferably about 150 nM of TSA; and about 2 μM to about 20 μM of 5-AZA, preferably about 5 μM. In one embodiment, the first stage of step (a) typically lasts about 1 to 2 days, for example about 1 day.

[0030] b) Second stage: The maintenance phase involves reducing the concentrations of DNA methyltransferase inhibitors and histone deacetylase inhibitors, while simultaneously inducing the targeted expression of neural stem cell markers using a small molecule combination of retinoic acid receptor agonists and GSK-3 inhibitors. In one specific embodiment of the second phase, the culture medium comprises neural basal medium as the basal medium and further contains 5-AZA, TSA, ATRA, and CHIR99021. In one embodiment, the culture medium contains approximately 10 nM to approximately 100 nM of TSA, preferably approximately 50 nM; approximately 0.2 μM to approximately 2 μM of 5-AZA, preferably approximately 1 μM; approximately 0.1 μM to approximately 10 μM of ATRA, preferably approximately 1 μM; and approximately 0.1 μM to approximately 10 μM of CHIR99021, preferably approximately 3 μM. In one embodiment, the second phase of step (b) typically lasts for approximately 2 to 4 days, for example, approximately 2 days.

[0031] c) Third stage: Adding a small molecule combination of retinoic acid receptor agonists and GSK-3 inhibitors to induce the expression of characteristic proteins PAX6, Nestine, β-III-Tubulin and / or SOX2; In one specific embodiment of the third stage, the culture medium comprises neural basal medium as the basal medium and further comprises ATRA and CHIR99021. In one embodiment, the culture medium contains about 0.1 μM to about 10 μM of ATRA, preferably about 1 μM; and about 0.1 μM to about 10 μM of CHIR99021, preferably about 3 μM; in one embodiment, the third stage of step (c) typically lasts about 5 to 8 days, for example about 5 days. Example

[0032] The subject matter of this disclosure will be better understood by referring to the following examples, which are provided as examples of the subject matter of this disclosure and are not intended to limit it. Information on the reagents used in the examples is listed in Table 1: Table 1 Reagent Name CAS number factory Item number Specification DMEM / F-12 none Gibco 21041025 500mL B27 none Azure Sky C0350 10mL N2 none Azure Sky C0335 25mL ITS none Genha R32316 10mL ascorbic acid 50-81-7 Genha S52732 100g EGF none novoprotein GMP-C029 500ug bFGF none Love Must Believe abs00877 10ug Trichostatin A 58880-19-6 Genha S48702 5mg 5-Zazacytidine 320-67-2 Genha B34297 100mg All-trans retinoic acid 302-79-4 Azure Sky ST1627 1g CHIR99021 252917-06-9 Sigma-Aldrich SML1046 5mg Example 1 In this embodiment, the neural basal culture medium consists of basal culture medium, nutrient factors and neurotrophic factors. The basal culture medium is DMEM / F12, the nutrient factors include 1% B27, 1% N2, 1% ITS and 300 mg / L ascorbic acid, and the neurotrophic factors are 50 ng / mL EGF and 20 ng / mL bFGF.

[0033] Phase 1: Human adipose-derived MSCs were obtained from human abdominal adipose fluid, digested with type I collagenase, and filtered through a cell strainer to obtain P0 generation cells. P1-P3 generation human adipose-derived MSCs were seeded into 6-well plates coated with a substrate gel. Once the cells adhered and reached 90% confluence, induction additives TSA and 5-AZA were added to the basal induction medium, and the cells were cultured for 24 hours. The concentration of TSA used in the first stage was 150 nM, and the concentration of 5-AZA was 5 μM.

[0034] Phase Two: The medium was replaced with a low concentration of TSA and 5-AZA and maintained for 48 hours. Induction additives ATRA and CHIR99021 were added. In the second stage, the concentration of induction additives TSA was 50 nM, 5-AZA was 1 μM, ATRA was 1 μM, and CHIR99021 was 3 μM.

[0035] Phase Three: After 48 hours, TSA and 5-AZA were removed from the second stage, and ATRA and CHIR99021 were added to the neural basal medium and cultured for 5 days. In the third stage, the concentration of the induction additive ATRA was 1 μM and the concentration of CHIR99021 was 3 μM.

[0036] Phase Four: Cells were digested by collagenase IV and trypsin, re-seeded in uncoated culture dishes, and spheroidized with neural basal culture medium. After stabilization for 48 hours, neural stem cells were obtained.

[0037] Example 2 The basal culture medium was the same as in Example 1.

[0038] Phase 1: ADMSCs were seeded in 6-well plates coated with substrate gel. After the cells adhered and reached 90% confluence, induction additives TSA and 5-AZA were added to the basal induction medium and cultured for 24 h. The concentration of induction additive TSA used in the first stage was 300 nM and the concentration of 5-AZA was 20 μM.

[0039] Phase Two: The medium was replaced with a low concentration of TSA and 5-AZA and maintained for 48 hours. Induction additives ATRA and CHIR99021 were added. In the second stage, the concentrations of induction additives TSA, 5-AZA, ATRA, and CHIR99021 were 10 μM.

[0040] Phase Three: After 48 hours, TSA and 5-AZA were removed from the second stage, and ATRA and CHIR99021 were added to the neural basal medium and cultured for 5 days. In the third stage, the concentration of the inducing additive ATRA was 10 μM and the concentration of CHIR99021 was 10 μM.

[0041] Phase Four: Cells were digested by collagenase IV and trypsin, re-seeded in uncoated culture dishes, and spheroidized with neural basal culture medium. After stabilization for 48 hours, neural stem cells were obtained.

[0042] Example 3 The basal culture medium was the same as in Example 1.

[0043] Phase 1: ADMSCs were seeded in 6-well plates coated with substrate gel. After the cells adhered and reached 90% confluence, induction additives TSA and 5-AZA were added to the basal induction medium and cultured for 24 h. The concentration of induction additive TSA in the first stage was 100 nM and the concentration of 5-AZA was 2 μM.

[0044] Phase Two: The medium was replaced with a low concentration of TSA and 5-AZA and maintained for 48 hours. Induction additives ATRA and CHIR99021 were added. In the second stage, the concentrations of induction additives TSA and 5-AZA were 10 nM, 5-AZA was 0.2 μM, ATRA was 0.1 μM, and CHIR99021 was 0.1 μM.

[0045] Phase Three: After 48 hours, TSA and 5-AZA were removed from the second stage, and ATRA and CHIR99021 were added to the neural basal medium and cultured for 5 days. In the third stage, the concentration of the inducing additive ATRA was 0.1 μM and the concentration of CHIR99021 was 0.1 μM.

[0046] Phase Four: Cells were digested by collagenase IV and trypsin, re-seeded in uncoated culture dishes, and spheroidized with neural basal culture medium. After stabilization for 48 hours, neural stem cells were obtained.

[0047] Comparative Example The neural basal culture medium consisted of basal medium, nutrient factors, and neurotrophic factors. The basal medium was DMEM / F12, the nutrient factors included 1% B27 and 1% N2, and the neurotrophic factors were 20 ng / mL EGF and 20 ng / mL bFGF. The culture period was 14 days. For specific operational steps to obtain neural stem cells, refer to the reference (Liu Jiaxin. Study on the therapeutic mechanism of induced neural stem cell transplantation on neuroinflammation caused by cerebral hemorrhage; Ningxia Medical University, 2023).

[0048] Identification of prepared neural stem cells Cell morphology Figure 1 The image shows a bright-field plot of NSCs obtained by targeted differentiation of ADMSCs according to the method in Example 1. Figure 1 (Left) shows the spherical state of the cells 12 hours after digestion and seeding, where the cells have begun to aggregate into clusters. Figure 1 (Right) The cells have formed well after 48 hours of inoculation. It can be seen that the NSCs obtained by the method provided by this invention have the typical morphology of NSCs.

[0049] Immunohistochemical staining Immunohistochemical staining was performed on the NSCs obtained in Example 1 to identify the positivity of characteristic NSC markers. The results are as follows: Figure 2 As shown, the characteristic indicators PAX6 (A), Nestine (B), and SOX2 (C) were significantly positive after DAB staining (brown), with a significantly higher positive rate than the control group (figure not shown).

[0050] Immunofluorescence staining The NSCs obtained from each protocol were subjected to β3-tubulin and Nestine immunofluorescence staining, and the results are as follows: Figure 3 (β3-tubulin) and Figure 4 As shown in (Nestine), 0# is the MSCs control group, 1# is the group using the patented protocol (Example 1), 2# is NSCs obtained using the conventional protocol (comparative example), 3# (Example 2), and 4# (Example 3). In the control group, both β3-tubulin and Nestine were negative. In Example 1, β3-tubulin expression was low, while Nestine showed significant positive expression, a characteristic of neural stem cells. Example 2 was a high-concentration induction protocol, where the expression of both β3-tubulin and Nestine became very weak, possibly related to cytotoxicity. In Example 3, β3-tubulin expression was low, while Nestine showed relatively obvious expression, but weaker than in Example 1. In the comparative example, β3-tubulin expression was high, and Nestine expression was also weak, indicating that the induction direction was towards neurons.

[0051] Those skilled in the art will further recognize that the invention can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of this disclosure only discloses exemplary embodiments, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein.

Claims

1. A method for in vitro culture of neural stem cells derived from mesenchymal stem cells, characterized in that, The method includes: (a) Mesenchymal stem cells were cultured in a medium containing HDAC inhibitors and DNA methyltransferase inhibitors to induce their differentiation, thereby forming cells that initiate the expression of neural stem cell marker genes; (b) The cells obtained in step (a) were cultured in a medium containing an HDAC inhibitor, a DNA methyltransferase inhibitor, a retinoic acid receptor agonist and a GSK-3 inhibitor to induce their differentiation, thereby forming cells that initiate the expression of neural stem cell marker proteins. (c) The cells obtained in step (b) are cultured in a medium containing a GSK-3 inhibitor and a retinoic acid receptor agonist to induce their differentiation, thereby forming neural stem cells.

2. The method according to claim 1, characterized in that, The concentration of the HDAC inhibitor in step (b) is lower than that in step (a); the concentration of the DNA methyltransferase inhibitor in step (b) is lower than that in step (a); and step (c) does not contain either the HDAC inhibitor or the DNA methyltransferase inhibitor.

3. The method according to claim 1, characterized in that, The HDAC inhibitor is one or more of trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entenolol (MS-275).

4. The method according to claim 1, characterized in that, The DNA methyltransferase inhibitor is one or more of 5-azacytidine (5-AZA), RG108, Decitabine, and SGI-1027.

5. The method according to claim 1, characterized in that, The retinoic acid receptor agonist is one or more of retinoic acid, AM580, all-trans retinoic acid (ATRA), TTNPB, and AC55649.

6. The method according to claim 1, characterized in that, The GSK-3 inhibitor is one or more of CHIR99021, BIP-135, CP21R7, 1-Azakenpaullone, TDZD-8, SB216763, SB415286, BIO-acetoxime, Tideglusib, and AR-A014418.

7. The method according to claim 1, characterized in that, The method further includes: (d) The neural stem cells obtained in step (c) are cultured in a neural basal medium to further mature and proliferate the neural stem cells.

8. A neural stem cell or a population thereof generated by or directly from the method of any one of claims 1-7.

9. A composition comprising the neural stem cells or cell populations thereof as described in claim 8.

10. Use of the neural stem cells or cell populations thereof as claimed in claim 8, or the composition as claimed in claim 9, in the preparation of a medicament for the treatment and / or prevention of neurodegenerative diseases.

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