Serum-free culture medium and application thereof in obtaining nerve cells by transdifferentiation of embryo fibroblasts

Abstract

The invention belongs to the technical field of biology, and discloses a serum-free culture medium and application thereof in obtaining nerve cells through embryo fibroblast transdifferentiation. The serum-free culture medium consists of a basic culture medium, a protein additive and cell culture auxiliary components, the protein additive consists of transferrin, a leukemia inhibition factor and a basic fibroblast growth factor; the final concentration of the transferrin is 5 ng / mL-30 ng / mL, the final concentration of the leukemia inhibition factor is 50 ng / mL-150 ng / mL, and the final concentration of the basic fibroblast growth factor is 10 ng / mL-25 ng / mL. The serum-free culture medium can promote the conversion speed of transdifferentiation of fibroblasts into nerve cells, can significantly improve the transdifferentiation efficiency, and can improve the cell survival rate of transdifferentiation of mouse embryo fibroblasts into nerve cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a serum-free culture medium and its application in the transdifferentiation of embryonic fibroblasts to obtain nerve cells. Background Technology

[0002] Neurological diseases, including neurodegenerative diseases, stroke, and spinal cord injury, have become a serious threat to human health. With the accelerating aging of society, the incidence of neurodegenerative diseases such as Alzheimer's and Parkinson's is rising year by year, placing a heavy burden on patients' families and society. Traditional treatments mainly focus on symptom relief but cannot fundamentally repair damaged neural networks. While regenerative medicine strategies such as neural stem cell transplantation offer new ideas, they face many challenges, such as limited cell sources, immune rejection, and ethical controversies. More importantly, neurons in the central nervous system of adult mammals are almost unable to self-renew after injury, making the repair of nervous system damage exceptionally difficult. Therefore, how to effectively replenish functional neurons and reconstruct damaged neural circuits has become a core scientific question in the fields of neuroscience and regenerative medicine.

[0003] Researchers have proposed various strategies for neuronal regeneration: activation of endogenous neural stem cells, transplantation of exogenous stem cells, and direct transdifferentiation of somatic cells. Among these, somatic transdifferentiation technology has attracted much attention due to its unique advantages. This technology refers to the process by which a terminally differentiated mature cell directly transforms into another functional cell type without going through a pluripotent intermediate state. In nature, there are numerous examples of this phenomenon; for instance, after lens damage in a newt, the pigment epithelial cells (PECs) on the dorsal side of the iris can transdifferentiate and regenerate the missing lens tissue. The concept of transdifferentiation was formally proposed in 1991, and the breakthrough in induced pluripotent stem cell (iPS) technology in 2006 provided a completely new approach to transdifferentiation across cell lineages. Initially, transdifferentiation research was limited to cell types within the same lineage or the same organ, but iPS technology has inspired researchers to explore the possibility of direct transformation between cells from different germ layers or even different lineages.

[0004] In 2008, Feng et al. achieved a significant breakthrough, discovering that overexpressing transcription factors C / EBPα and PU.1 in fibroblasts could transdifferentiate them into macrophages with phagocytic function. This demonstrated that even between distantly related somatic cells, cell fate can be altered by regulating key transcription factors. This discovery inspired researchers to explore transdifferentiation of other cell types, particularly the transdifferentiation of neurons, which are difficult to regenerate. In 2010, Vierbuchen et al. achieved a landmark breakthrough, successfully reprogramming mouse fibroblasts directly into electrophysiologically active neuron-like cells for the first time by overexpressing three transcription factors: Ascl1, Brn2, and Myt1l (collectively known as the BAM combination). These induced neurons (iN cells) not only expressed neuron-specific markers but also formed synaptic connections and generated action potentials. Just one year later, multiple laboratories successively achieved transdifferentiation from human fibroblasts to neurons, marking a new stage in neural transdifferentiation technology.

[0005] From a mechanistic perspective, cell fate transition depends on the precise coordination of transcription factor networks and epigenetic regulation. In 2013, Wapinski et al. revealed the phased mechanism of transdifferentiation mediated by BAM factors: Ascl1, as a "pioneer transcription factor," first recognizes and binds to closed chromatin regions in fibroblasts, particularly regulatory sites of neuron-specific genes; subsequently, it recruits Brn2 to these sites; while Myt1l mainly functions in the later maturation stage. Notably, Zfp238 was identified as a key downstream target gene of Ascl1, crucial for neuron fate determination. This finding highlights the importance of precise matching between pioneer factors and the chromatin background. Further research shows that transcription factor expression can be transient and does not need to be sustained throughout the transition, overcoming the potential side effects and functional interference of permanent overexpression of neurodevelopmental transcription factors.

[0006] Besides transcription factors, small molecule compounds also play an important role in neural transdifferentiation. Studies have shown that inhibiting the TGFβ signaling pathway (e.g., using Noggin or ALK inhibitors) can significantly improve transdifferentiation efficiency. In 2015, research found that small molecule combinations can convert fibroblasts into functional neurons; these combinations typically include GSK3β inhibitors, ALK inhibitors, and cAMP enhancers. These chemical induction methods provide new tools for optimizing the transdifferentiation process, but transdifferentiation efficiency and neuronal functional maturity still need improvement.

[0007] However, neural transdifferentiation technology still faces significant challenges. First, transdifferentiation efficiency is generally low. Vierbuchen et al. reported that even under optimized conditions, only about 20% of mouse embryonic fibroblasts successfully transdifferentiated into functional neurons. Second, the obtained neurons often exhibit immature characteristics in morphology and marker protein expression, making it difficult to fully mimic the properties of natural neurons. Third, traditional serum-containing culture media have complex compositions and large batch-to-batch variations; unknown factors in serum may interfere with transcription factor-mediated fate determination processes. The applicant of this invention has successfully improved the transdifferentiation efficiency of fibroblasts into cardiomyocytes and hepatocytes by developing a serum-free culture medium with a specific formulation, demonstrating the feasibility and significant application value of this research direction. Furthermore, optimizing the culture system is a key factor in improving transdifferentiation efficiency, but existing transdifferentiation schemes still have low efficiency in achieving neuronal transdifferentiation and limited effects on promoting cell survival and functional maturity. There is an urgent need to develop more optimized serum-free culture systems to fully unleash the potential of somatic cell transdifferentiation in the field of neural regeneration. Summary of the Invention

[0008] The purpose of this invention is to provide a serum-free culture medium that can promote the rate and efficiency of fibroblast transdifferentiation into nerve cells, as well as improve the survival rate of transdifferentiated cells.

[0009] The following technical solutions are used to achieve the above objectives.

[0010] The first aspect of the present invention provides a serum-free culture medium, which is composed of a basal culture medium, a protein additive, and cell culture auxiliary components;

[0011] The protein additive is composed of transferrin, leukemia inhibitory factor, and basic fibroblast growth factor.

[0012] The final concentration of the transferrin is 5 ng / mL to 30 ng / mL, the final concentration of the leukemia inhibitory factor is 50 ng / mL to 150 ng / mL, and the final concentration of the basic fibroblast growth factor is 10 ng / mL to 25 ng / mL.

[0013] In some embodiments, the final concentration of the transferrin is 5 ng / mL to 7 ng / mL, the final concentration of the leukemia inhibitory factor is 90 ng / mL to 110 ng / mL, and the final concentration of the basic fibroblast growth factor is 15 ng / mL to 25 ng / mL.

[0014] In some embodiments, the final concentration of the transferrin is 5.2 ng / mL to 6 ng / mL, the final concentration of the leukemia inhibitory factor is 95 ng / mL to 105 ng / mL, and the final concentration of the basic fibroblast growth factor is 17 ng / mL to 23 ng / mL.

[0015] Preferably, the final concentration of the transferrin is 5.5±0.1 ng / mL, the final concentration of the leukemia inhibitory factor is 100±1 ng / mL, and the final concentration of the basic fibroblast growth factor is 20±1 ng / mL.

[0016] In some embodiments, the cell culture aid is L-glutamine; the final concentration of the cell culture aid is 1 mM to 10 mM.

[0017] In some embodiments, the final concentration of the cell culture aid is 1 mM to 5 mM, preferably 1.5 mM to 2.5 mM.

[0018] In some embodiments, the basal culture medium is at least one of DMEM, MEM, BME, F-10, F-12, RPMI1640, GMEM, αMEM, and IMDM.

[0019] In some embodiments, the basal culture medium is a mixture of DMEM and F12; preferably, the volume ratio of DMEM to F12 in the basal culture medium is 1:1.

[0020] A second aspect of the present invention provides the application of the serum-free culture medium described above in the direct transdifferentiation of embryonic fibroblasts into nerve cells.

[0021] In some embodiments, the embryonic fibroblasts are mouse embryonic fibroblasts or human embryonic fibroblasts, preferably mouse embryonic fibroblasts.

[0022] A third aspect of the present invention provides a method for transdifferentiating embryonic fibroblasts into nerve cells, comprising the following steps:

[0023] (a) Preparation of embryonic fibroblasts;

[0024] (b) Culture embryonic fibroblasts in the serum-free medium as described above for 2-3 days under cell growth conditions suitable for embryonic fibroblasts;

[0025] (c) Using platE cells to package retroviruses overexpressing Ascl1, Brn2, and Myt1l, the retroviruses were added to cells cultured in serum-free medium for infection, and the cells were then cultured in neural medium to obtain neural cells.

[0026] In this invention, a novel serum-free culture medium is obtained by adding specific protein additives and cell culture aids to the basic cell culture medium. Before viral infection of embryonic fibroblasts, mouse embryonic fibroblasts are cultured using this novel serum-free culture medium. Through specific combinations of the culture medium and appropriate concentration control, the rate of fibroblast transdifferentiation into nerve cells can be promoted, and the transdifferentiation efficiency can be significantly improved. In addition, the cell survival rate of mouse embryonic fibroblasts that transdifferentiate into nerve cells can be increased, providing technical support for the future application of nerve cells in the treatment of clinical diseases. Attached Figure Description

[0027] Figure 1 These are morphological changes in cells obtained after culturing in serum-containing and serum-free media.

[0028] Figure 2 This is a graph showing the cell count results after culturing cells with and without serum-containing culture media.

[0029] Figure 3 This is a schematic diagram showing the results of cell expression of the neuronal marker (Tuj1) after culturing with and without serum-containing culture media.

[0030] Figure 4 This is a schematic diagram showing the effect of serum-free culture medium treatment time on the number of mouse embryonic fibroblasts expressing the neuronal marker (Tuj1).

[0031] Figure 5 This is a schematic diagram of the flow cytometry sorting results of continuous culture in serum-free medium on the expression of the neuronal marker (Tuj1) in mouse embryonic fibroblasts.

[0032] Figure 6 This is a schematic diagram of the immunoblotting results of different serum-free culture media on the expression of the neural cell marker (Tuj1) in mouse embryonic fibroblasts.

[0033] Figure 7 This is a schematic diagram of the flow cytometry sorting results of different serum-free culture media on the expression of the neuronal marker (Tuj1) in mouse embryonic fibroblasts.

[0034] Figure 8This is a schematic diagram of the flow cytometry sorting results of different concentrations of serum-free culture medium on the expression of the neuronal marker (Tuj1) in mouse embryonic fibroblasts. Detailed Implementation

[0035] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0036] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0038] In this invention, a novel serum-free culture medium is obtained by adding specific protein additives and cell culture aids to a basic cell culture medium. Before using viral overexpression of exogenous transcription factors (Ascl1, Brn2, Myt1l), culturing mouse embryonic fibroblasts in this novel serum-free medium promotes the transdifferentiation of mouse embryonic fibroblasts into neurons. The resulting neurons express the neuron-specific marker protein Tuj1. This method combines exogenous transcription factors with protein additives, and by setting appropriate concentrations and combinations of culture media, it can accelerate the transdifferentiation of fibroblasts into neurons, significantly improve transdifferentiation efficiency, and increase the survival rate of mouse embryonic fibroblasts transdifferentiated into neurons. This method also mitigates the risk of tumorigenesis to some extent.

[0039] To facilitate understanding of this technology, some terms and phrases are defined below.

[0040] "Basal medium" refers to any culture medium capable of supporting cell growth. Basal media that can be used in this invention include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. In a preferred embodiment, the basal medium is a mixture of DMEM and F12. In a more preferred embodiment, the basal medium is a 1:1 volume ratio mixture of DMEM and F12.

[0041] "Protein additives" refer to additives used in cell culture, added to the basal culture medium to support cell survival and growth. These factors are generally not included in the basal culture medium but are provided by the serum commonly used for cell culture. The protein additive used in this article is a mixed additive obtained by mixing transferrin, leukemia inhibitory factor, and basic fibroblast growth factor in a certain proportion.

[0042] The virus used in this invention was prepared using conventional techniques known to those skilled in the art, see, for example, Morita S et al., Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 2000;7(12):1063-1066.

[0043] The “suitable conditions for cell growth” described in this invention refer to conventional stem cell culture conditions in the art, and include some modifications suitable for specific cell lines but without affecting the basic properties of the cells. For culture methods and conditions, please refer to W. French Anderson et al., HANDBOOK OF STEM CELLS, Volume 2.

[0044] The method for detecting nerve cells described in this invention is well known to those skilled in the art; see, for example, Ferreira, A., & Caceres, A. (1992). Expression of the class III beta-tubulinisotype in developing neurons in culture. Journal of neuroscience research, 32(4), 516–529.

[0045] Unless otherwise specified, all reagents mentioned in this article are from Invitrogen, Sigma, or Merck Chemical Technology (Shanghai) Co., Ltd.

[0046] The present invention will be further described in detail below with reference to specific embodiments.

[0047] Example 1: The serum-free culture medium described in this invention improves the transdifferentiation efficiency of mouse embryonic fibroblasts into nerve cells.

[0048] I. Isolation and Culture of Mouse Embryonic Fibroblasts (MEF)

[0049] Mice 13–14 days of gestation were selected as donors and euthanized by cervical dislocation. The carcasses were then disinfected by immersing in 0.5% benzalkonium chloride solution for 30 seconds. After removal, they were placed on a sterile dissecting tray (lined with absorbent paper) and fixed abdominally. The abdominal skin was wiped with 75% ethanol to reduce the risk of hair contamination. Using sterile surgical instruments, the skin and muscle layers were sequentially incised to expose the abdominal cavity. Instruments were changed to avoid contamination. Fat and connective tissue attached to the uterus were peeled off, and the uterus was quickly removed and transferred to a 10 cm culture dish pre-containing PBS containing double antibiotics.

[0050] Inside a laminar flow hood, the uterine tissue was washed three times with PBS containing antibiotics. The amniotic sac was cut open with sterile ophthalmic scissors, the embryo was separated, and transferred to another culture dish containing antibiotic-containing PBS. The embryo was cut into 1–3 mm pieces. 3 Tissue blocks of a certain size were added to a mixture of 0.25% and 0.05% trypsin solution and digested at 37°C for 10 minutes until the suspension became turbid. An equal volume of MEF medium (high glucose DMEM, 10% FBS, 1× GlutaMAX, 1× NEAA) was added to stop the reaction. The cells were gently pipetted 5–10 times to disperse them. After standing for a few moments, the suspension was transferred to a 50 mL centrifuge tube and centrifuged at 200×g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in MEF medium. The pellet was seeded into 15 cm culture dishes, the medium was added to the required volume, and the mixture was stirred well. The dishes were then incubated at 37°C in a 5% CO2 incubator.

[0051] The following day, cell adhesion was observed, the culture medium was replaced with fresh medium, and the cells cultured this time were recorded as generation P0. The MEF medium was prepared as follows (500 mL): 430 mL high glucose DMEM, 60 mL fetal bovine serum (FBS), 5 mL GlutaMax, and 5 mL non-essential amino acids (NEAA).

[0052] Once the cells have grown to a suitable density, aspirate the culture medium and wash once with DPBS. Add an appropriate amount of 0.25% trypsin and digest at 37°C for 1–2 minutes. Add an equal volume of MEF medium to stop the digestion. Use a Pasteur pipette to detach the cells and mix well. Transfer to a 15 mL centrifuge tube and centrifuge at 200×g for 5 minutes. Discard the supernatant; use a portion of the cell suspension for cell counting, and centrifuge the remainder.

[0053] Prepare cryopreservation solution: 10% DMSO + 90% FBS. Resuspend cells in the cryopreservation solution and adjust the concentration to 3 × 10⁻⁶ cells / mL. 6 Cells / mL, aliquot 0.5 mL into each tube. Label with cell name, passage number, and cryopreservation date. Place the cryopreservation tubes in a programmed cooling box and incubate overnight at -80°C. Transfer to liquid nitrogen the following day for long-term storage.

[0054] II. Retrovirus Packaging and Collection

[0055] Plat-E packaging cells were revived and expanded, and transfected when the cell confluence reached approximately 80%. Plat-E cells are a highly efficient retroviral packaging system that can produce high-titer viruses in a short time after transfection with a specific plasmid (for preparation methods, see S. Morita et al., Plat-E: an efficient and stable system for transient packaging of retroviruses).

[0056] One hour before transfection, replace the Plat-E cell culture medium with fresh medium. Dissolve the plasmids in 1 mL of Opti-MEM at a standard rate of 10 μg plasmid DNA (pMMLV-Ascl1, pMMLV-Brn2, pMMLV-Myt1l) per 10 cm culture dish, vortex vigorously, and incubate at room temperature for 5 minutes. Then, add 40 μL of polyethyleneimine (PEI) per mL, vortex vigorously, and incubate for 15 minutes to form the transfection complex. Add the mixture to the Plat-E cell culture dish, gently shake to mix, and incubate at 37°C.

[0057] Replace with fresh culture medium after 12 hours. Collect viral supernatant at 36 and 60 hours post-transfection. For the first collection, aspirate the culture medium into a syringe, filter through a 0.45 μm filter membrane, and transfer to a centrifuge tube, storing temporarily at an appropriate temperature. Simultaneously, replenish with fresh culture medium to maintain cell condition for the second viral collection.

[0058] III. Transdifferentiation of Mouse Embryonic Fibroblasts (MEF)

[0059] Remove the frozen MEF cell tubes from liquid nitrogen and thaw them rapidly in a 37°C water bath, gently agitating them to accelerate the thawing process. After thawing, wipe the outer wall dry with sterile absorbent paper and disinfect the surface of the cryovials with 75% ethanol before transferring them to a biosafety cabinet. Slowly add the cell suspension to a 15 mL centrifuge tube containing 6 mL of MEF medium (high glucose DMEM, 10% FBS, 1× GlutaMAX, 1× NEAA), diluting dropwise with gentle agitation to avoid damaging the cells due to sudden changes in osmotic pressure. Centrifuge at 200×g for 5 minutes, discard the supernatant, resuspend the cell pellet in an appropriate amount of MEF medium, seed them into tissue culture dishes, gently agitate to ensure even cell distribution, and incubate statically at 37°C in a 5% CO2 incubator.

[0060] When cells have adhered and grown to approximately 80% confluence, they are passaged. The original culture medium is aspirated, and the cells are washed once with DPBS buffer. 1 mL of 0.25% trypsin solution is added, and the cells are incubated at 37°C for 3 minutes for digestion. When microscopic observation shows cell edge retraction and increased intercellular spaces, 2 mL of MEF medium is added to terminate the reaction. Cells are repeatedly pipetted to detach completely and form a single-cell suspension. This suspension is transferred to a 15 mL centrifuge tube and centrifuged at 200×g for 5 minutes. The supernatant is discarded. A portion of the cells is used for cell counting, and the remaining cells are adjusted to the desired concentration and seeded into 24-well plates at 1×10⁶ cells per well. 4 100 cells. Once the cells have fully adhered (usually 12–24 hours), begin serum-free culture treatment.

[0061] The serum-free culture medium is based on an equal volume mixture of DMEM and F12 medium, with the following components added: transferrin at a final concentration of 5.5 ng / mL, basic fibroblast growth factor (bFGF) at 20 ng / mL, leukemia inhibitory factor at 100 ng / mL, and L-glutamine at 2 mM. All components are added directly to the basal medium and mixed thoroughly by inverting until fully dissolved, taking care to avoid vigorous shaking that generates excessive foam. The prepared serum-free culture, stored at 4°C in the dark, can maintain stable activity for up to two weeks. Protein additives (such as transferrin and bFGF) should be stored at -20°C according to the product instructions. They can be pre-prepared as high-concentration stock solutions, aliquoted, and frozen to avoid repeated freeze-thaw cycles that could lead to inactivation. Small molecule compounds should first be prepared as high-concentration stock solutions from powder, aliquoted separately, and stored at -20°C.

[0062] Cells were cultured in serum-free medium for 0-3 days before retroviral infection. A two-round consecutive infection strategy was employed. First, retroviral supernatant containing the target genes (Ascl1, Brn2, Myt1l) was added, and after 12 hours of infection, the medium was replaced with fresh MEF medium for further culture. A second round of infection was then performed after 12 hours, with the addition of viral solution for another 12 hours. After both infections, the virus-containing medium was completely removed and replaced with neural cell induction medium, formulated as follows: DMEM / F12, 25 g / mL insulin, 50 g / mL transferrin, 30 nM sodium selenite, 20 nM progesterone, and 100 nM putrescine. This day was defined as day 0 (D0).

[0063] Cell morphology changes were observed daily, and bright-field images were taken to record the cell remodeling process. The first day after viral infection ended was designated D0, and cells were continuously cultured until D16, with cell status and medium changes recorded throughout the process. Before each change to serum-free medium, the medium was brought to room temperature to equilibrate and prevent thermal shock from affecting cell status.

[0064] IV. Immunofluorescence analysis of cell samples

[0065] Pretreated circular coverslips were placed in a clean bench and briefly sterilized by igniting residual anhydrous ethanol with an alcohol lamp. The coverslips were then placed in 24-well cell culture plates, and Matrigel was added to cover the surface. The plates were incubated overnight at 37°C to promote matrix solidification. MEF cells were then seeded onto the coverslips and cultured and induced as described above.

[0066] At time point D16, at the end of the induction culture, remove the corresponding well plate, discard the culture medium, gently wash the cells once with pre-warmed or room temperature PBS, then add 4% paraformaldehyde fixative and fix at room temperature for 30 minutes. After fixation, rinse three times with PBS for 5 minutes each time to thoroughly remove residual fixative.

[0067] Next, cell membrane permeabilization was performed: 0.3% Triton X-100 (dissolved in PBS) was added, and the cells were incubated at room temperature for 20 minutes. The cells were then washed three times with PBS, 5 minutes each time. To reduce non-specific binding, the cells were blocked for 2 hours at room temperature using PBS containing 3% bovine serum albumin (BSA). After blocking, the cells were washed three more times with PBS, 5 minutes each time.

[0068] Dilute the primary antibody with PBS containing 1% BSA according to the dilution ratio recommended in the antibody instructions. Add 200 μL to each well and incubate overnight at 4°C. The next day, remove the solution and wash away any unbound primary antibody with PBS three times, for 5 minutes each time.

[0069] Then, 200 μL of fluorescently labeled secondary antibody (Invitrogen, diluted 1:400 with PBS) was added to each well, and the mixture was incubated at room temperature for 1 hour in the dark. After incubation, the mixture was washed three times with PBS for 5 minutes each time to remove unbound secondary antibody.

[0070] Finally, perform nuclear staining: add DAPI staining solution (final concentration 1 μg / mL, prepared with PBS), and stain at room temperature in the dark for 5 minutes. After staining, wash three times with PBS for 5 minutes each time to remove excess dye. Remove the coverslip from the well plate, drain the liquid, and then fix it onto the slide with mounting medium.

[0071] After sample preparation, the samples were observed under an inverted fluorescence microscope, and images were acquired and results recorded. The expression status of the target protein was determined by the fluorescence signal, and the transdifferentiation efficiency was evaluated.

[0072] The control group was the treatment without the addition of serum-free medium (no treatment was performed before viral infection, and the culture was routinely carried out in MEF medium).

[0073] The results are as follows Figure 1As shown, mouse embryonic fibroblasts were cultured in serum-free medium for two days before viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l. Immediately after viral infection, the morphological changes of the obtained cells were observed using bright-field microscopy. The results showed that, compared to the control group (which was cultured in conventional MEF medium without viral infection but without viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l), cells treated with serum-free medium for two days exhibited cell shrinkage and a tendency towards neural processes, while the control group remained in the typical morphology of mouse embryonic fibroblasts. This indicates that adding serum-free medium for two days before viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l can significantly increase the rate of transdifferentiation of mouse embryonic fibroblasts into neural cells.

[0074] like Figure 2 As shown, compared to the control group without serum-free medium, the total number of cells obtained after retrovirus-mediated overexpression of Ascl1, Brn2, and Myt1l, combined with pretreatment with serum-free medium and subsequent neural transdifferentiation induction culture, was significantly higher. Since the initial cell counts in the experimental and control groups were identical before serum-free medium treatment, the cell counting results indicate that culturing with serum-free medium for 2 days significantly improves the cell survival rate of mouse embryonic fibroblasts transdifferentiating into neural cells.

[0075] like Figure 3 As shown, compared to the control group without serum-free culture medium, the cell population treated with retrovirus-mediated overexpression of Ascl1, Brn2, and Myt1l, combined with pretreatment with serum-free culture medium and subsequent specific induction culture, exhibited positive expression of the neural lineage marker Tuj1, indicating the generation of cells with neuronal characteristics. This result demonstrates that this culture system can effectively promote the transdifferentiation of mouse embryonic fibroblasts into neurons.

[0076] Example 2: Improving the efficiency of transdifferentiation of mouse embryonic fibroblasts into neural cells using serum-free continuous culture medium.

[0077] Before using retroviruses to overexpress exogenous transcription factors Ascl1, Brn2, and Myt1l, mouse embryonic fibroblasts were continuously cultured for 0, 1, 2, or 3 days using serum-free medium prepared in Example 1. After viral infection, the medium was replaced with neural cell induction medium and cultured until D16 (16 days). The expression of the neural cell marker Tuj1 was detected by immunofluorescence and Western blotting.

[0078] like Figure 4As shown, mouse embryonic fibroblasts were continuously cultured in serum-free medium for 1-3 days before viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l. After viral infection, the medium was replaced with neuronal induction medium and cultured until day 16 (16 days). Cells were then fixed with 4% paraformaldehyde, and the expression of the neuronal marker Tuj1 in the obtained cells was detected using immunofluorescence. The results showed that the obtained cells could express the neuronal marker Tuj1. Compared with the control group (which used viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l but did not use serum-free medium, but were cultured in conventional MEF medium before viral infection), continuous culture in serum-free medium, especially after 2 days of treatment, significantly increased the number of green fluorescently labeled Tuj1-positive cells in the transdifferentiated cells, and the neurite length was significantly prolonged. This indicates that adding serum-free medium for continuous culture for 2 days before viral overexpression of exogenous transcription factors Ascl1, Brn2, and Myt1l can significantly improve the efficiency and quality of transdifferentiation of mouse embryonic fibroblasts into neural cells.

[0079] like Figure 5 As shown, mouse embryonic fibroblasts were continuously cultured in serum-free medium for 1-3 days, followed by a neuronal transdifferentiation program. After 16 days, the cells were fixed with 4% paraformaldehyde, and the expression of the neuronal marker Tuj1 in the obtained cells was quantitatively analyzed by flow cytometry. The results showed that the obtained cells could express the neuronal marker Tuj1. Compared with the control group (using MEF medium but not pre-treated with serum-free medium for viral overexpression of exogenous transcription factors) and the blank group (using DMEM / F12 medium without any small molecules for pre-treatment with exogenous transcription factors), the proportion of Tuj1-positive cells reached 33.8%±6.5% during continuous culture in serum-free medium, especially at day 2, which was significantly higher than the 20.3%±3.1% in the control group (p<0.01). This result further confirms that continuous culture in serum-free medium for 2 days before virus-mediated transcription factor overexpression can significantly improve the efficiency of transdifferentiation of mouse embryonic fibroblasts into neurons. In summary, this embodiment demonstrates that the treatment protocol of adding serum-free medium for two consecutive days before using retroviruses to overexpress neuron-specific transcription factors Ascl1, Brn2, and Myt1l can maximize the transdifferentiation of mouse embryonic fibroblasts into functional neurons, providing an efficient and reliable cell acquisition method for neuroregenerative medicine research.

[0080] Example 3: Promoting the transdifferentiation of mouse embryonic fibroblasts into nerve cells using different serum-free culture media

[0081] Before using the virus to overexpress exogenous transcription factors Ascl1, Brn2, and Myt1l, different serum-free culture media (including media based on a 1:1 volume ratio of DMEM and F12 mixed medium, supplemented with different concentrations and combinations of transferrin, leukemia inhibitory factor, basic fibroblast growth factor, and L-glutamine) were used. Mouse embryonic fibroblasts were first cultured for 2 days in different serum-free media, then the exogenous transcription factors were overexpressed using the virus, and the cells were cultured in a different neural medium until day 16. Immunofluorescence was used to determine the proportion of cells expressing the neural cell marker (Tuj1).

[0082] The composition of the serum-free culture medium is shown in Table 1 below:

[0083] Table 1 Composition of serum-free culture medium

[0084]

[0085] The results are as follows Figures 6-7 As shown in the figure, group 1 corresponds to the control group in the figure. "+" in the figure means that the corresponding component was added, and "-" means that the corresponding component was not added.

[0086] like Figure 6 As shown, before the exogenous transcription factor was overexpressed using the virus, the treatment group with serum-free medium containing transferrin, leukemia inhibitory factor, basic fibroblast growth factor, and L-glutamine (corresponding to group 6 in the table) had the highest protein levels of the neuronal marker (Tuj1) compared to the control group using only MEF medium. This indicates that the cells induced by the serum-free medium expressed more neuronal markers.

[0087] like Figure 7 As shown, before the exogenous transcription factor was overexpressed using the virus, compared with the control group that only used MEF medium, the serum-free medium containing transferrin, leukemia inhibitory factor, basic fibroblast growth factor, and L-glutamine (corresponding to group 6 in the table) had the highest proportion of cells expressing the neuronal marker (Tuj1) and the highest transdifferentiation efficiency. Although other groups of serum-free medium could also obtain cells expressing the neuronal marker (Tuj1), the transdifferentiation efficiency was not high. This indicates that only the serum-free medium containing transferrin, leukemia inhibitory factor, basic fibroblast growth factor, and L-glutamine has the most significant effect on the transdifferentiation of mouse embryonic fibroblasts into neural cells.

[0088] Example 4: Effects of serum-free culture medium with different component concentrations on the transdifferentiation efficiency of mouse embryonic fibroblasts into neurons.

[0089] Before using the virus to overexpress exogenous transcription factors Ascl1, Brn2, and Myt1l, different serum-free culture media (including media based on a 1:1 volume ratio of DMEM and F12 mixed medium, supplemented with different concentrations and combinations of transferrin, leukemia inhibitory factor, basic fibroblast growth factor, and L-glutamine) were used. Mouse embryonic fibroblasts were first cultured for 2 days in different serum-free media, then overexpressed exogenous transcription factors Ascl1, Brn2, and Myt1l using the virus, and the cells were cultured in a different neural medium until day 16. Immunofluorescence was used to determine the proportion of cells expressing the neural cell marker (Tuj1).

[0090] The composition of the serum-free culture medium is shown in Table 2 below:

[0091] Table 2 Composition of serum-free culture medium

[0092]

[0093] The results are as follows Figure 8 As shown, the treatment without serum-free medium served as the control group (directly cultured in MEF medium). Changes in the concentrations of transferrin, leukemia inhibitory factor, and basic fibroblast growth factor all affected transdifferentiation efficiency. Before using the virus to overexpress exogenous transcription factors, compared with the control group, the transdifferentiation efficiency in the serum-free medium treatment group with a final concentration of 5 ng / mL for transferrin, 100 ng / mL for leukemia inhibitory factor, and 20 ng / mL for basic fibroblast growth factor was significantly higher than that in the control group. Although other concentrations of serum-free medium could also improve the transdifferentiation efficiency of embryonic fibroblasts into nerve cells to some extent, the improvement effect was not significant. This indicates that only the serum-free medium with a combination of the three concentrations of transferrin, leukemia inhibitory factor, and basic fibroblast growth factor has the most significant effect on the transdifferentiation of mouse embryonic fibroblasts into nerve cells.

[0094] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A serum-free culture medium, characterized in that, The serum-free culture medium consists of a basal culture medium, protein additives, and cell culture auxiliary components. The protein additive is composed of transferrin, leukemia inhibitory factor, and basic fibroblast growth factor. The final concentration of the transferrin is 5 ng / mL to 30 ng / mL, the final concentration of the leukemia inhibitory factor is 50 ng / mL to 150 ng / mL, and the final concentration of the basic fibroblast growth factor is 10 ng / mL to 25 ng / mL.

2. The serum-free culture medium as described in claim 1, characterized in that, The final concentration of the transferrin is 5 ng / mL to 7 ng / mL, the final concentration of the leukemia inhibitory factor is 90 ng / mL to 110 ng / mL, and the final concentration of the basic fibroblast growth factor is 15 ng / mL to 25 ng / mL.

3. The serum-free culture medium as described in claim 2, characterized in that, The final concentration of the transferrin is 5.5±0.1 ng / mL, the final concentration of the leukemia inhibitory factor is 100±1 ng / mL, and the final concentration of the basic fibroblast growth factor is 20±1 ng / mL.

4. The serum-free culture medium according to any one of claims 1-3, characterized in that, The cell culture aid is L-glutamine; the final concentration of the cell culture aid is 1 mM to 10 mM.

5. The serum-free culture medium as described in claim 4, characterized in that, The final concentration of the cell culture auxiliary component is 1 mM to 5 mM, preferably 1.5 mM to 2.5 mM.

6. The serum-free culture medium according to any one of claims 1-3, characterized in that, The basal culture medium is at least one of DMEM, MEM, BME, F-10, F-12, RPMI 1640, GMEM, αMEM, and IMDM.

7. The serum-free culture medium as described in claim 6, characterized in that, The basal culture medium is a mixture of DMEM and F12; preferably, the volume ratio of DMEM to F12 in the basal culture medium is 1:

1.

8. The use of the serum-free culture medium according to any one of claims 1-7 in the direct transdifferentiation of embryonic fibroblasts into nerve cells.

9. The application as described in claim 8, characterized in that, The embryonic fibroblasts are mouse embryonic fibroblasts or human embryonic fibroblasts, preferably mouse embryonic fibroblasts.

10. A method for transdifferentiating embryonic fibroblasts into nerve cells, characterized in that, Includes the following steps: (a) Preparation of embryonic fibroblasts; (b) Embryonic fibroblasts are cultured using the serum-free culture medium according to any one of claims 1-7 for 2-3 days under cell growth conditions suitable for embryonic fibroblasts; (c) Using platE cells to package retroviruses overexpressing Ascl1, Brn2, and Myt1l, the retroviruses were added to cells cultured in serum-free medium for infection, and the cells were then cultured in neural medium to obtain neural cells.

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