Method for inducing and converting mesenchymal cells into neural stem cells

Abstract

The invention discloses a method for inducing and converting mesenchymal cells into neural stem cells, which relates to the technical field of biology and comprises the following steps: (a) providing a group of mesenchymal stem cells cultured in vitro; (b) culturing the mesenchymal stem cells in an induction medium, and inducing the mesenchymal stem cells to differentiate into neural stem cells; wherein the induction medium comprises a basal medium and the following components in effective induction concentrations: a histone deacetylase inhibitor (HDAC inhibitor); a glycogen synthase kinase-3beta inhibitor (GSK-3beta inhibitor); a retinoic acid receptor agonist; the invention provides a set of technical scheme for inducing and transforming the mesenchymal stem cells into the neural stem cells for the first time, which is clear in components, efficient, stable, high in universality and simple and convenient to operate, not only fills the blank in the prior art, but also provides a new cell source and technical support for medical research and clinical treatment of nerve regeneration. The method has an important scientific research value and a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for inducing mesenchymal cells to transform into neural stem cells. Background Technology

[0002] Neural stem cells (NSCs) possess self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into neurons, astrocytes, and oligodendrocytes, showing broad application prospects in areas such as neurodegenerative disease model construction, drug screening, damage repair, and regenerative medicine. However, traditional methods of obtaining NSCs mainly rely on embryonic or adult neural tissue, which is not only subject to ethical constraints but also suffers from limited sources, insufficient expansion capacity, and immune rejection, severely limiting their basic research and clinical translation.

[0003] To overcome the limitations of stem cell sources, induced pluripotent stem cell (iPSC) technology has been widely explored. However, its induction process is complex and time-consuming, and it carries risks such as tumorigenicity and genetic instability, limiting its clinical application. Therefore, researchers have gradually turned their attention to somatic cell direct reprogramming technology, attempting to obtain neural stem cells through a safer and more efficient approach.

[0004] Mesenchymal stem cells (MSCs) have become an important cell source in regenerative medicine due to their ease of isolation from various tissues such as bone marrow, adipose tissue, and umbilical cord, as well as their good in vitro expansion capacity and low immunogenicity. Early studies attempted to guide MSCs to differentiate into neural cells using single growth factors (such as bFGF and EGF) or chemical inducers, but these often only induced morphological changes or transient expression of some neural marker proteins, failing to obtain true neural stem cells with self-renewal capacity and multilineage differentiation potential. Furthermore, the induction efficiency of MSCs from different tissue sources varies significantly, and the reproducibility of the system is poor, which also limits the practical application of this strategy.

[0005] Currently, although studies have attempted to combine various small molecule compounds and growth factors to improve induction efficiency, a standardized induction protocol with clearly defined components, high induction efficiency, good stability, and applicability to MSCs from different sources is still lacking. Therefore, developing a method to efficiently and stably convert human MSCs into functional neural stem cells is not only helpful in solving the cell source problem but also of great significance for promoting cell therapy for neurological diseases.

[0006] Current technologies for inducing neural stem cells from MSCs still suffer from low efficiency, incomplete function, and poor system universality. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for inducing mesenchymal cells to transform into neural stem cells, solving the technical problems of low efficiency, incomplete function, and poor system universality in the induction of MSCs into neural stem cells in existing technologies.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for inducing mesenchymal stem cells to transform into neural stem cells, comprising the following steps: (a) Provide a population of in vitro cultured mesenchymal stem cells; (b) The mesenchymal stem cells were cultured in an induction medium to induce their differentiation into neural stem cells; The induction medium comprises a basal medium and the following components at an effective induction concentration: Histone deacetylase inhibitors (HDAC inhibitors); Glycogen synthase kinase-3β inhibitors (GSK-3β inhibitors); retinoic acid receptor agonists; Neurotrophic factors; (c) Maintain culture for 5-21 days, changing the induction medium every 2-4 days; (d) Obtain a cell population with self-renewal capacity and multi-directional neural differentiation potential.

[0009] Preferably, the HDAC inhibitor is valproic acid (VPA) or a pharmaceutically acceptable salt thereof, at a concentration of 0.5-2 mM; The GSK-3β inhibitor is CHIR99021, with a concentration of 3-10 μM; The retinoic acid receptor agonist is all-trans retinoic acid (ATRA) at a concentration of 0.1-5 μM; The neurotrophic factors are basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), both at a concentration of 10-50 ng / mL. Preferably, the basal culture medium is a 1:1 mixture of DMEM / F12 and Neurobasal medium, supplemented with B27 additive, N2 additive, non-essential amino acids and antioxidants.

[0010] Preferably, the mesenchymal stem cells described in step (a) are of human origin and are selected from bone marrow, adipose tissue, Wharton's jelly from the umbilical cord, or dental pulp.

[0011] Preferably, the culture conditions in step (b) are: 37°C, 5% CO2 saturated humidity environment, and low-adhesion culture dishes or culture dishes coated with matrix gel are used during the induction process.

[0012] Preferably, the cell population in step (d) highly expresses neural stem cell markers Nestin, Sox2, and Pax6, and does not express or expresses low levels of mesenchymal stem cell markers CD90 and CD105.

[0013] Preferably, step (d) is followed by functional validation of the obtained neural stem cells: (e) The cells are cultured in a differentiation medium to differentiate into neurons, astrocytes, and oligodendrocytes; (f) The expression of neuronal markers (β-III-tubulin, MAP2), astrocyte markers (GFAP), and oligodendrocyte markers (O4, MBP) was detected by immunofluorescence staining or RT-PCR.

[0014] Beneficial effects This invention provides a method for inducing mesenchymal cells (MSCs) to transform into neural stem cells, solving the technical problems of low efficiency, incomplete function, and poor system universality in the induction of MSCs into neural stem cells in the prior art. This invention has the following advantages: 1. This invention is the first to employ a multi-factor synergistic combination of histone deacetylase inhibitors (HDAC inhibitors), glycogen synthase kinase-3β inhibitors (GSK-3β inhibitors), retinoic acid receptor agonists, and neurotrophic factors, significantly improving induction efficiency and cell reprogramming stability. Through the synergistic effects of valproic acid (VPA), CHIR99021, all-trans retinoic acid (ATRA), and bFGF and EGF, human mesenchymal stem cells can be efficiently induced into cell clusters with typical neurosphere structures in a short period of time (5–21 days), and the positive expression rate of neural stem cell markers (Nestin, Sox2, Pax6) exceeds 80%, far higher than that of traditional single growth factor induction methods (usually less than 15%).

[0015] 2. The induction system established in this invention has clearly defined components, optimized concentration range, and good reproducibility. It is applicable to human mesenchymal stem cells from different tissue sources, including adipose tissue, bone marrow, Wharton's jelly from the umbilical cord, and dental pulp, demonstrating good universality and stability. Examples show that neural stem cells can be successfully obtained from both human adipose-derived MSCs and umbilical cord-derived MSCs using the same induction protocol, greatly expanding the cell sources and application scope.

[0016] 3. The cell population obtained in this invention not only highly expresses neural stem cell markers but also possesses self-renewal capacity and multi-lineage differentiation potential. In vitro differentiation verification showed that these cells can further differentiate into neurons (expressing β-III-tubulin and MAP2), astrocytes (expressing GFAP), and oligodendrocytes (expressing O4 and MBP), indicating that they possess true functional neural stem cell characteristics and provide a reliable cell source for establishing neurological disease models, drug screening, and cell transplantation therapy.

[0017] 4. The induction strategy employed in this invention avoids genetic manipulations such as viral vectors or gene transfection, relying entirely on small molecule compounds and growth factors to achieve cell fate conversion. This approach is simple to operate, highly safe, and better meets clinical application requirements. Furthermore, the inducers used, such as VPA, CHIR99021, and ATRA, are commonly used and commercially mature compounds in research, with controllable costs, facilitating widespread adoption and large-scale culture.

[0018] 5. By setting up a comparative example (using only bFGF and EGF), it was confirmed that the factor combination used in this invention is non-obvious and synergistically necessary, with each factor being indispensable. This combination not only significantly improves the induction efficiency but also maintains long-term stable cell proliferation, avoiding early apoptosis or non-directed differentiation, thus solving the technical bottlenecks of incomplete induction and unstable cell state in existing technologies.

[0019] This invention is the first to propose a technical solution for the induction and transformation of mesenchymal stem cells into neural stem cells that is characterized by clear composition, high efficiency and stability, strong universality and simple operation. It not only fills the gap in existing technology, but also provides new cell sources and technical support for neural regeneration medicine research and clinical treatment, and has important scientific research value and broad application prospects. Detailed Implementation

[0020] The present invention will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] Example 1: Induction of neural stem cells from human adipose-derived mesenchymal stem cells (hAD-MSCs) 1. Material preparation Cell source: Commercially purchased or self-isolated and cultured human adipose-derived MSCs (passages 3-5).

[0022] Basal medium: DMEM / F12 and Neurobasal medium mixed 1:1.

[0023] Additives: B27Supplement (50×), N2Supplement (100×), NEAA (100×), β-mercaptoethanol (55mM).

[0024] Inducing factors: Valproic acid (VPA, Sigma) was prepared as a 500 mM stock solution using PBS.

[0025] CHIR99021 (Selleck) was prepared as a 10 mM stock solution using DMSO.

[0026] All-trans retinoic acid (ATRA, Sigma) was prepared into a 10 mM stock solution using DMSO. Human recombinant bFGF (PeproTech) was prepared into a 20 μg / mL stock solution using PBS containing 0.1% BSA.

[0027] Human recombinant EGF (PeproTech) was prepared into a 20 μg / mL stock solution using PBS containing 0.1% BSA.

[0028] Complete induction medium: Add the following components to the basal medium to the final concentration: 1×B27, 1×N2, 0.1mM NEAA, 0.1mM β-mercaptoethanol 1mMVPA, 5μM CHIR99021, 1μM ATRA, 20ng / mLbFGF, 20ng / mLEGF.

[0029] Culture vessel: 6-well plate with ultra-low adsorption (Corning).

[0030] 2. Induction process (1) Take hAD-MSCs with good growth status and fusion degree of 80%-90%, digest them with 0.25% trypsin and resuspend them into a single cell suspension.

[0031] (2) After counting, use 5 × 10 4 cells / cm 2 The culture medium was seeded at a density of 100% in 6-well ultra-low adsorption plates, and 2 mL of complete induction medium was added to each well.

[0032] (3) Place the cells in a 37°C, 5% CO2 incubator. Replace half of the total induction medium with fresh complete induction medium every 3 days: gently collect the supernatant of the old medium, centrifuge (1000 rpm, 5 min), discard half of the supernatant, resuspend the cell pellet with an equal volume of fresh complete induction medium, and return it to the original well for continued culture.

[0033] (4) After culturing for about 7-10 days, a large number of regularly shaped cell clusters (neurospheres) with suspended growth can be observed under a microscope.

[0034] 3. Identification and Testing (1) Identification of NSCs markers by immunofluorescence staining: Collect neurospheres, dissociate them into single cells using Accutase, and seed them into Matrigel-coated 24-well plates.

[0035] After the cells adhered to the membrane, they were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA.

[0036] Add primary antibodies (mouse anti-human Nestin monoclonal antibody, 1:200; rabbit anti-human Sox2 polyclonal antibody, 1:200) and incubate overnight at 4°C.

[0037] After washing with PBS, add the corresponding fluorescently labeled secondary antibodies (Alexa Fluor 488 labeled goat anti-mouse IgG, 1:500; Alexa Fluor 594 labeled goat anti-rabbit IgG, 1:500) and incubate at room temperature in the dark for 1 hour.

[0038] Cell nuclei were counterstained with DAPI and observed under a fluorescence microscope. The vast majority of cells were Nestin and Sox2 positive (>80%), confirming successful induction into NSCs.

[0039] (2) RT-PCR detection of gene expression: Total RNA was extracted from the induced cells and reverse transcribed into cDNA.

[0040] PCR amplification was performed using specific primers.

[0041] The results showed that the induced cells highly expressed NSCs-related genes (Nestin, Sox2, Pax6), while the expression of MSCs marker genes (CD90, CD105) was significantly downregulated or disappeared.

[0042] (3) Verification of multi-directional differentiation potential: The obtained neurospheres were dissociated and seeded into poly-L-lysine-coated culture plates, which were then replaced with neurobasal medium (Neurobasal + B27 + 1% FBS + 10 ng / mL BDNF).

[0043] After 14 days of culture, immunofluorescence staining was performed.

[0044] Differentiated cells expressed the neuronal marker β-III-tubulin and the astrocyte marker GFAP. O4 and MBP-positive cells were also detected when using a culture medium for directed oligodendrocyte differentiation. This demonstrates that the induced NSCs possess the ability to differentiate into three neural lineages.

[0045] Example 2: Induction of neural stem cells from human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs) Except for the cell source being changed to MSCs derived from Wharton's jelly in human umbilical cord, the other induction steps, culture medium formulation, and identification methods were the same as in Example 1.

[0046] Neurosphere formation was also observed after 7-10 days of culture. Immunofluorescence and RT-PCR confirmed that the induced cells highly expressed Nestin and Sox2 and could differentiate into neurons and glial cells in differentiation medium, demonstrating that the method of this invention is universal and effective for human MSCs from different sources.

[0047] Comparative example: Single growth factor induction hAD-MSCs were cultured under the same conditions using a medium containing only bFGF (20 ng / mL) and EGF (20 ng / mL) (excluding VPA, CHIR99021, ATRA).

[0048] Cell morphological changes were not obvious, and neurospheres were rarely formed. Immunofluorescence assays showed that the proportion of Nestin-positive cells was extremely low (<15%), and the cells were difficult to passage for a long time, quickly undergoing apoptosis or differentiation, making it impossible to obtain functional NSCs. This comparison demonstrates the non-obviousness and synergistic necessity of the inducing factor combination of the present invention.

[0049] It should be noted that in this paper, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

Claims

1. A method for inducing mesenchymal stem cells to transform into neural stem cells, characterized in that, Includes the following steps: (a) Provide a population of in vitro cultured mesenchymal stem cells; (b) The mesenchymal stem cells were cultured in an induction medium to induce their differentiation into neural stem cells; The induction medium comprises a basal medium and the following components at an effective induction concentration: Histone deacetylase inhibitors (HDAC inhibitors); Glycogen synthase kinase-3β inhibitors (GSK-3β inhibitors); retinoic acid receptor agonists; Neurotrophic factors; (c) Maintain culture for 5-21 days, changing the induction medium every 2-4 days; (d) Obtain a cell population with self-renewal capacity and multi-directional neural differentiation potential.

2. The method for inducing mesenchymal cells to transform into neural stem cells according to claim 1, characterized in that... The HDAC inhibitor is valproic acid (VPA) or a pharmaceutically acceptable salt thereof, at a concentration of 0.5-2 mM; The GSK-3β inhibitor is CHIR99021, with a concentration of 3-10 μM; The retinoic acid receptor agonist is all-trans retinoic acid (ATRA) at a concentration of 0.1-5 μM; The neurotrophic factors are basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), both at a concentration of 10-50 ng / mL.

3. A method for inducing mesenchymal cells to transform into neural stem cells according to any one of claims 1 or 2, characterized in that... The basal culture medium is a 1:1 mixture of DMEM / F12 and Neurobasal medium, supplemented with B27 additive, N2 additive, non-essential amino acids and antioxidants.

4. The method for inducing mesenchymal cells to transform into neural stem cells according to claim 1, characterized in that... The mesenchymal stem cells mentioned in step (a) are of human origin and are selected from one of bone marrow, adipose tissue, Wharton's jelly from the umbilical cord, or dental pulp.

5. The method for inducing mesenchymal cells to transform into neural stem cells according to claim 1, characterized in that... The culture conditions in step (b) are: 37°C, 5% CO2 saturated humidity environment, and low-adhesion culture dishes or culture vessels coated with matrix gel are used during the induction process.

6. The method for inducing mesenchymal cells to transform into neural stem cells according to claim 1, characterized in that... The cell population described in step (d) highly expresses neural stem cell markers Nestin, Sox2, and Pax6, and does not express or expresses low levels of mesenchymal stem cell markers CD90 and CD105.

7. The method for inducing mesenchymal cells to transform into neural stem cells according to claim 1, characterized in that... Step (d) is followed by functional validation of the obtained neural stem cells: (e) The cells are cultured in a differentiation medium to differentiate into neurons, astrocytes, and oligodendrocytes; (f) The expression of neuronal markers (β-III-tubulin, MAP2), astrocyte markers (GFAP), and oligodendrocyte markers (O4, MBP) was detected by immunofluorescence staining or RT-PCR.