Methods for direct transdifferentiation of primordial germ cells into neural stem cell-like cells

Abstract

The present application provides a method for directly transdifferentiating spermatogonial stem cells (SSCs) into neural stem cell-like cells (iNSCs). The iNSCs have proliferative activity, can be stably subcultured in vitro, and have the potential to differentiate into other neural cells such as neurons, astrocytes and oligodendrocytes. The method of the present application has high efficiency, can obtain more iNSCs in a relatively short period in vitro, and can obtain iNSCs with high purity, wherein the double positive rate of Nestin and Pax6 is more than 95%.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, and more specifically, this invention relates to a method for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells. Background Technology

[0002] Neurological diseases are characterized by high recurrence rates, high prevalence, and significant harm. It is estimated that the global prevalence of neurological diseases is approximately 5%. Common neurological diseases include Alzheimer's disease, epilepsy, spinal cord injury, astrocytoma, and oligodendroma. However, the occurrence of these diseases is closely related to the loss or abnormality of function in neurons, astrocytes, or oligodendrocytes. Neural stem cells (NSCs) are cells capable of self-renewal and proliferation, and can differentiate into different types of functional neurons, astrocytes, and oligodendrocytes. These cells play a crucial role in maintaining the normal function of the body's nervous system. Therefore, NSCs have broad application prospects in basic research and clinical treatment of neurological diseases; however, the limited number of NSCs in vivo restricts their clinical application. Therefore, cell source is one of the key issues that must be addressed to accelerate the clinical application of NSCs. Currently, NSCs can be obtained through direct isolation and extraction from primitive tissues and differentiation from pluripotent stem cells. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), possess multipotent differentiation potential and are an effective alternative to primary cell separation. Although researchers are exploring the direct differentiation of ESCs and iPSCs into neural stem cell-like cells (iNSCs), ethical concerns regarding ESC sourcing and safety concerns regarding iPSC sourcing limit their clinical application.

[0003] Transdifferentiation refers to the process of reprogramming differentiated cells of one type to transform them into another type of differentiated cell in terms of structure and function. This process is mainly induced by exogenously expressed cell-specific transcription factors and compounds. Current research indicates that transient expression of pluripotency factors combined with appropriate neural signal input or direct overexpression of NSC-specific transcription factors Sox2 or ZFP521 can directly transdifferentiate human and mouse fibroblasts into iNSCs. Nevertheless, the expression of exogenous genes involved in this process is usually mediated by lentiviruses, which makes its safety in clinical applications uncertain.

[0004] Therefore, there is an urgent need in this field to find a direct transdifferentiation method that does not involve lentiviruses or transgenic manipulation, in order to provide an effective way to overcome the above-mentioned limiting factors. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells.

[0006] In a first aspect of the present invention, a method for preparing neural stem cell-like cells is provided, comprising: using spermatogonial stem cells as starting cells, directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells.

[0007] In one or more preferred embodiments, the method for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells includes:

[0008] (1) Culturing spermatogonial stem cells in spermatogonial stem cell culture medium;

[0009] (2) The cells cultured in (1) are cultured in transdifferentiation medium A; wherein the transdifferentiation medium A includes spermatogonial stem cell culture medium and retinoic acid;

[0010] (3) The cells cultured in (2) are cultured in transdifferentiation medium B; the transdifferentiation medium B includes: a mixture of complete medium and neural medium, and N2B27, serum albumin, insulin, SB431542, L-glutamine, β-mercaptoethanol, serum, and bFGF; to obtain neural stem cell-like cells.

[0011] In one or more preferred embodiments, the method further includes: (4) culturing or passaged the cells cultured in (3) in an amplification medium; wherein the amplification medium is based on transdifferentiation medium B, in which serum is replaced with EGF.

[0012] In one or more preferred embodiments, spermatogonial stem cells are isolated from testicular tissue using a two-step enzymatic digestion method.

[0013] In one or more preferred embodiments, in (2), spermatogonial stem cells are seeded in a pre-coated container (e.g., a well plate); preferably, the container is coated with adhesion protein (10±5 μg / mL, more preferably 10±3 μg / mL or 10±2 μg / mL); preferably, 5000 to 20000 cells / well are seeded in a 24-well plate; preferably, the plate is cultured at 37±2°C.

[0014] In one or more preferred embodiments, in (2), the culture time is 24 ± 12 hours; more preferably 24 ± 8 hours; more preferably 24 ± 6 hours (e.g., 24 ± 4 hours, 24 ± 3 hours, 24 ± 2 hours, etc.).

[0015] In one or more preferred embodiments, in (3), the culture time is more than 2 days; preferably 3 to 8 days; more preferably 3.5 to 7 days (e.g., 4, 5, 6 days).

[0016] In one or more preferred embodiments, the spermatogonial stem cell culture medium comprises: a basal culture medium, and GDNF, EGF, bFGF, LIF, non-essential amino acids, L-glutamine, bovine insulin, vitamins, transferrin, putrescine, progesterone, β-mercaptoethanol, and serum; preferably, it comprises:

[0017]

[0018] Preferably, the basal culture medium is MEM-α medium.

[0019] In some embodiments, the vitamin may be a finished product with a concentration of 100×, which is adjusted to a working concentration of 1× before use.

[0020] In one or more preferred embodiments, the transdifferentiation culture medium B comprises:

[0021]

[0022] Preferably, the mixed medium of complete culture medium and nerve culture medium is mixed at a volume ratio of 1:5 to 5:1 (preferably 1:3 to 3:1, more preferably 1:2 to 2:1); more preferably, the complete culture medium is DMEM / F12 medium and the nerve culture medium is Neurobasal medium.

[0023] In one or more preferred embodiments, in (2), the amount of retinoic acid is 0.2 to 5 μM (preferably 0.4 to 3 μM, more preferably 0.7 to 1.5 μM, such as 1 ± 0.2 μM).

[0024] In one or more preferred embodiments, in (4), the amount of EGF is 5 to 100 ng / mL (preferably 8 to 60 ng / mL, more preferably 12 to 40, such as 20 ± 5 ng / mL or 20 ± 3 ng / mL).

[0025] In one or more preferred embodiments, in (1), the spermatogonial stem cells obtained by culture are cultured in an STO cell feeder layer for transdifferentiation.

[0026] In one or more preferred embodiments, after using the transdifferentiation medium, 20±5 ng / mL (preferably 20±3 ng / mL or 20±2 ng / mL) of bFGF is added daily.

[0027] In one or more preferred embodiments, (1) the cultured spermatogonial stem cells (highly) express MVH, PLZF , OCT4 , ETV5 , GFRα1 .

[0028] In one or more preferred embodiments, in (3), the cultured neural stem cell-like cells (highly) express Vimentin , ID2 , Nrcam, Nestin , Blbp , CD133 , Cdh2 , L1cam Low expression or no expression Plzf, Id4, Nanos2, Nanos3, Neurog2, Sholh1, Bcl6b, Etv5, Oct4 .

[0029] In one or more preferred embodiments, in (3), the cultured neural stem cell-like cells are Nestin and Pax6 positive.

[0030] In another aspect of the present invention, a method for preparing nerve cells is provided, comprising:

[0031] (a) Obtain neural stem cell-like cells or passaged cells thereof by any of the preceding methods;

[0032] (b) Further induce the differentiation of the cells in (a) into nerve cells; said nerve cells include: neurons, astrocytes, oligodendrocytes.

[0033] In one or more preferred embodiments, the nerve cells are neurons, cultured using a neuron differentiation medium; preferably, the neuron differentiation medium comprises: a mixed medium of complete medium and neuron medium, and N2, B27, L-glutamine, anti-Hesl oligonucleotide chain, and 3-isobutyl-1-methylxanthine.

[0034] In one or more preferred embodiments, the nerve cells are astrocytes, cultured using an astrocyte differentiation medium; preferably, the astrocyte differentiation medium includes: a complete culture medium, and non-essential amino acids, L-glutamine, and fetal bovine serum.

[0035] In one or more preferred embodiments, the nerve cells are oligodendrocytes, cultured using an oligodendrocyte differentiation medium; preferably, the oligodendrocytes comprise: a complete culture medium and N2,3-isobutyl-1-methylxanthine.

[0036] In one or more preferred embodiments, the neuronal differentiation culture medium comprises:

[0037]

[0038] In one or more preferred embodiments, the astrocyte differentiation culture medium comprises:

[0039]

[0040] In one or more preferred embodiments, the oligodendrocyte differentiation culture medium comprises:

[0041]

[0042] In one or more preferred embodiments, the cultured astrocytes express (high) [expression of...]. Gfap , S100β .

[0043] In one or more preferred embodiments, the cultured oligodendrocytes (highly express) Ng2, Olig2, Mbp wait.

[0044] In one or more preferred embodiments, the cultured neurons exhibit (high) expression NSE, Tuj1, NeuN, Map2, Gad65, Gad67 .

[0045] In one or more preferred embodiments, the cultured neurons are positive for MAP2, Tuj1, GAD65, and GAD67.

[0046] In one or more preferred embodiments, the cultured neurons have the ability to generate action potentials.

[0047] In another aspect of the present invention, a neural stem cell-like cell is provided, which is obtained directly from spermatogonial stem cells through transdifferentiation.

[0048] In one or more preferred embodiments, the neural stem cell-like cells express neural stem cell markers Nestin and Pax6; more preferably, the double positivity rate of Nestin and Pax6 reaches more than 95%.

[0049] In one or more preferred embodiments, the neural stem cell-like cells are obtained by the method described.

[0050] In another aspect of the invention, an application is provided for any of the methods described above, for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells; or for preparing neural cells, said neural cells comprising: neurons, astrocytes, oligodendrocytes.

[0051] In one or more embodiments, the above methods and applications are in vitro methods and applications (non-therapeutic, non-diagnostic), and the cells comprise: cell cultures; or, the culture is carried out in a container.

[0052] In another aspect of the invention, a kit is provided for the in vitro preparation of neural stem cell-like cells or neural cells differentiated therefrom, comprising:

[0053] Spermatogonial stem cell culture medium; preferably, comprising: basal culture medium, and GDNF, EGF, bFGF, LIF, non-essential amino acids, L-glutamine, bovine insulin, vitamins, transferrin, putrescine, progesterone, β-mercaptoethanol, and serum;

[0054] Transdifferentiation medium A; the transdifferentiation medium A includes spermatogonial stem cell culture medium and retinoic acid;

[0055] Transdifferentiation medium B; the transdifferentiation medium B includes: a mixed medium of complete medium and nerve medium, and N2B27, serum albumin, insulin, SB431542, L-glutamine, β-mercaptoethanol, serum, and bFGF;

[0056] Preferably, the kit further includes an amplification medium; more preferably, the amplification medium is based on transdifferentiation medium B, with serum replaced by EGF;

[0057] Preferably, the kit further includes a neuronal differentiation culture medium; more preferably, the neuronal differentiation culture medium comprises: a mixed culture medium of complete culture medium and neuronal culture medium, and N2, B27, L-glutamine, anti-Hesl oligonucleotide chain, and 3-isobutyl-1-methylxanthine;

[0058] Preferably, the kit further includes an astrocyte differentiation medium; more preferably, the astrocyte differentiation medium comprises: a complete medium, and non-essential amino acids, L-glutamine, and fetal bovine serum;

[0059] Preferably, the kit further includes an oligodendrocyte differentiation culture medium; more preferably, the oligodendrocytes comprise: a complete culture medium and N2,3-isobutyl-1-methylxanthine.

[0060] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0061] Figure 1 SSCs identification;

[0062] (A) MVH immunofluorescence staining of SSCs; Scale bar, 20 μm;

[0063] (B) Immunofluorescence staining of SSCs with PLZF and OCT4; Scale bar, 50 μm;

[0064] (C) RT-PCR detection Plzf , Gfrα1 , Etv5 , Mvh and Oct4 Expression in SSCs and STO cells.

[0065] Figure 2 SSCs directly transform into iNSCs;

[0066] (A) Cell morphological changes during transdifferentiation of SSCs to iNSCs; Scale bar, 20 μm;

[0067] (B) qRT-PCR was used to detect the expression of NSCs-related genes during transdifferentiation;

[0068] (C) qRT-PCR was used to detect the expression of SSCs markers and related genes during transdifferentiation;

[0069] (D) Nestin and Pax6 immunofluorescence staining of cells on day 6 of transdifferentiation; Scale bar, 20 μm.

[0070] Figure 3 Analysis of the proliferation capacity of iNSCs;

[0071] (A) Nestin, Pax6 and Ki67 immunofluorescence staining of P1 generation iNSCs; Scale bar, 50 μm;

[0072] (B) Nestin, Pax6 and Ki67 immunofluorescence staining of P5 generation iNSCs; Scale bar, 50 μm;

[0073] (C) Nestin, Pax6 and Ki67 immunofluorescence staining of P10 generation iNSCs; Scale bar, 50 μm;

[0074] (D) Statistical results of Nestin and Pax6 double positivity rates of iNSCs with different culture generations.

[0075] Figure 4 The potential for iNSCs to differentiate into astrocytes;

[0076] (A) Cell morphology of iNSCs 4 weeks after differentiation into astrocytes; Scale bar, 50 μm;

[0077] (B) RT-PCR detection of astrocytes derived from iNSCs Gfap and S100β Gene expression.

[0078] Figure 5 The differentiation potential of iNSCs into oligodendrocytes;

[0079] (A) Cell morphology of iNSCs after differentiation into oligodendrocytes; Scale bar, 50 μm;

[0080] (B) RT-PCR detection of oligodendrocytes derived from iNSCs Ng2,Olig2 and Mbp Gene expression.

[0081] Figure 6 The potential for iNSCs to differentiate into neurons;

[0082] (A) Cell morphology of iNSCs 3 weeks after differentiation into neurons; Scale bar, 50 μm;

[0083] (B) RT-PCR was used to detect the expression of neuronal markers in iNSCs and differentiated neurons;

[0084] (C)qRT-PCR was used to detect the expression of neuronal markers at 1 week, 2 weeks and 3 weeks after iNSCs differentiated into neurons.

[0085] (D) Immunofluorescence staining of MAP2 and Tuj1, and MAP2 and GAD65+67, in neurons differentiated from iNSCs. Scale bar, 50 μm;

[0086] (E) Electrophysiological detection of iNSCs 3 weeks after differentiation into neurons; the left figure shows the neurons for electrophysiological detection, the middle figure shows sodium and potassium ion currents, and the right figure shows action potentials.

[0087] Figure 7 The differentiation potential of iNSCs in the brain;

[0088] (A) Immunofluorescence staining of mcherry and eGFP in paraffin sections of mouse brain tissue 4 weeks after iNSC injection. White arrows indicate mcherry-positive and eGFP-positive cells, and yellow arrows indicate mcherry-positive and eGFP-negative cells. Scale bar, 50 μm;

[0089] (B) Statistical results of the number of mcherry-positive cells in paraffin sections of whole brain tissue of mice 4 weeks after injection of iNSCs;

[0090] (C) Statistical results of the proportion of eGFP positive cells and eGFP negative cells in mcherry positive cells 4 weeks after injection of iNSCs;

[0091] (D) Immunofluorescence staining of mcherry, Tuj1, and GAD65+67 in paraffin sections of mouse brain tissue 4 weeks after iNSC injection. White arrows indicate that the injected iNSCs differentiated into neurons in the brain. Scale bar, 50 μm;

[0092] (E) Statistical results of the proportion of GAD65 / 67 positive cells and GAD65 / 67 negative cells among mcherry positive cells and eGFP negative cells 4 weeks after iNSC injection. Detailed Implementation

[0093] Through in-depth research, the inventors have revealed a highly efficient method for directly transdifferentiating spermatogonial stem cells (SSCs) into neural stem cell-like cells (iNSCs). These iNSCs exhibit proliferative activity, can be stably passaged in vitro, and possess the potential to differentiate into other neural cells such as neurons, astrocytes, and oligodendrocytes. The method of this invention provides a holistic optimized approach that is highly efficient, enabling the acquisition of a large number of iNSCs in vitro within a very short period, and yielding highly pure iNSCs with a Nestin and Pax6 double positivity rate exceeding 95%, which is beneficial for downstream cell isolation and purification.

[0094] SSCs (Seminal Stem Cells) are a type of adult stem cell capable of passing on genetic information to the next generation. In the testicular microenvironment, SSCs can self-renew and differentiate into sperm to maintain male fertility. When the microenvironment of SSCs changes, they will no longer differentiate into sperm following a strict spermatogenesis process, but will instead transdifferentiate into other cell types based on their current microenvironment. Current research has found that SSCs can be directly transdifferentiated into hepatocytes, kidney cells, and neurons using in vitro culture systems and tissue culture techniques, but there are no reports of SSCs directly transdifferentiating into iNSCs (Induced Noncellular Root Cells).

[0095] The inventors of this invention have focused on the in vitro preparation of functional neural stem cell-like cells and have revealed a method for differentiating spermatogonial stem cells into neural stem cell-like cells. In a preferred embodiment, this method induces spermatogonial stem cells to differentiate in vitro by rationally designing a transdifferentiation stage and adding key factors that induce differentiation, thereby obtaining functional neural stem cell-like cells.

[0096] As used in this invention, "functional" means that the neural stem cell-like cells obtained according to the described methods have the same or similar functions as expected.

[0097] As used in this invention, "differentiation" refers to the developmental process of lineage commitment. "Lineage" refers to the pathway of cell development.

[0098] Based on the inventor's new discovery, this invention provides a method for preparing neural stem cell-like cells in vitro, comprising: (1) culturing spermatogonial stem cells in spermatogonial stem cell culture medium; (2) culturing the cells cultured in (1) in transdifferentiation culture medium A; wherein transdifferentiation culture medium A includes spermatogonial stem cell culture medium and retinoic acid; (3) culturing the cells cultured in (2) in transdifferentiation culture medium B; thereby obtaining neural stem cell-like cells. During the culture period, fresh culture medium may be used as needed to replace the culture medium.

[0099] In this invention, the method described is applicable to the transdifferentiation of spermatogonial stem cells derived from mammals. The animals include non-human mammals or humans, preferably including (but not limited to): rodents (including mice, rats, hamsters, etc.), non-human primates (such as monkeys, orangutans, etc.), and livestock (such as cattle, sheep, dogs, pigs, rabbits, etc.).

[0100] Unless otherwise stated, the culture medium used for culturing or induction is a liquid culture medium (culture solution).

[0101] In this invention, the spermatogonial stem cells can be spermatogonial stem cells derived from the body or from spermatogonial stem cells that have already been expanded, cultured, passaged, or established. For example, the inventors have already established spermatogonial stem cells in previous studies. For example, a method for preparing spermatogonial stem cells includes: isolating and preparing spermatogonial stem cells from testicular tissue using a two-step enzymatic digestion method (type IV collagenase + trypsin).

[0102] In a specific embodiment of the present invention, the transdifferentiation of SSCs to iNSCs is carried out through the following specific steps: (1) Spermatogonial stem cells are isolated using a two-step enzymatic digestion method and expanded in vitro using SSCs culture medium; (2) Approximately 10,000 SSCs / well are seeded in a 24-well plate coated with adhesions and cultured in vitro at 37°C for 24 hours, followed by the addition of 1 μM retinoic acid to the SSCs culture medium and continued culturing for another 24 hours; (3) The culture medium is replaced with transdifferentiation medium and cultured at 37°C for 6 days. It should be understood that this is a preferred example, and the overall scheme of the present invention is not limited thereto.

[0103] The main features and advantages of the method of this invention are as follows: First, the entire culture system uses cell growth factors and chemical molecules, without introducing exogenous genes involved in reprogramming or transdifferentiation (and without altering the genome structure). Therefore, it avoids interference with the genome stability of the original stem cells by exogenous genes and the transplantation safety risks associated with exogenous cells. Second, it can efficiently obtain neural stem cell-like cells with Nestin and Pax6 double positivity. Third, the efficiently obtained neural stem cell-like cells have a high transdifferentiation efficiency, ideal cell state, and can well support the further differentiation of neural stem cell-like cells into neural cells.

[0104] The transdifferentiation method of the present invention can obtain high-purity iNSCs in vitro, with a double positivity rate of Nestin and Pax6 exceeding 95%.

[0105] The transdifferentiation method of the present invention is an orderly and holistic approach that requires very little time and is highly efficient (e.g., only 8 days).

[0106] Meanwhile, through optimized design of the culture program and culture medium, the transdifferentiation method of this invention does not require going through a pluripotency stage (such as...). Figure 2 Results C show that the pluripotency gene Oct4 Instead of relying on gene expression levels to decrease during transdifferentiation, direct adherent transdifferentiation culture can be achieved, which is simple to operate and has a high success rate.

[0107] The neural stem cell-like cells obtained by this invention have multiple applications, including further induction to differentiate into various types of nerve cells (including neurons, astrocytes, oligodendrocytes, etc.), achieving efficient differentiation and obtaining nerve cells with typical characteristics. Furthermore, these nerve cells can form brain cell tissue.

[0108] The neural stem cell-like cells obtained by this invention can be enriched at the site of injury and differentiate into nerve cells in the local tissue / microenvironment, repairing and replenishing damaged nerve cells. For example, ischemia and hypoxia can cause such injury, and the neural stem cell-like cells are enriched at the site of injury. These neural stem cell-like cells can also stimulate existing neurons and glial cells by producing various neurotrophic factors, promoting the repair of damaged cells. Furthermore, these neural stem cell-like cells can enhance the connections between neural synapses, establish new neural circuits, and reduce oxidative stress in the brain.

[0109] The neural stem cell-like cells described in this invention can be applied to a variety of central nervous system diseases, including brain and spinal cord injuries. Examples include, but are not limited to: cerebral palsy, meningitis or its sequelae, brain dysplasia, stroke (cerebral hemorrhage or cerebral infarction) or its sequelae, traumatic brain injury, spinal cord injury, motor neuron disease, amyotrophic lateral sclerosis (ALS), brain atrophy, ataxia, Parkinson's disease, epilepsy, multiple system atrophy, Alzheimer's disease or vascular dementia, chorea, polyradiculitis, sensorineural hearing loss, facial paralysis, peripheral neuropathy, etc.

[0110] The present invention also provides culture media for various stages of induction culture, including the SSCs culture medium, transdifferentiation medium A, and transdifferentiation medium B, said culture media being usable for culturing SSCs cells and for generating iNSCs. Preferably, it is used as the iNSCs amplification culture medium.

[0111] In addition to the specific cytokines or chemical components listed in the embodiments of this invention, cytokines or chemical components known in the art that have the same or similar functions may also be used in this invention. Analogs, homofunctional proteins (such as homofunctional proteins of growth factors), or compounds of the specifically listed components, equivalent compounds inducing the same target, analogs, derivatives, and / or their salts, hydrates, or precursors may also be used to replace the specifically listed components to achieve the same technical effect. These analogs, homofunctional proteins, or compounds should also be included in this invention. Analogs of compounds include, but are not limited to, isomers and racemates of compounds. Compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis, or trans isomers. The term "precursor of a compound" refers to a compound, when applied or treated by appropriate methods, that can be converted in a culture medium into any of the above-mentioned compounds, or a salt or solution of any of the above-mentioned compounds.

[0112] As a preferred embodiment of the invention, the culture medium may also contain components for preventing bacterial contamination of cell culture, such as Gram-positive and Gram-negative bacterial contamination, for example, some antibiotics. In a preferred embodiment, double antibiotics are used.

[0113] The cytokines or chemical components are added to a suitable basal / complete / neural medium or mixed medium. The basal / complete medium may be MEM-α, DMEM / F12, DMEM, RPMI 1640, or an alternative medium with similar nutritional components; the neural medium may be Neuronal basal or an alternative medium with similar nutritional components. It should be understood that those skilled in the art are familiar with the preparation or purchase of the aforementioned basal cell culture media. Preferred cell culture media are provided in the embodiments of the present invention.

[0114] The present invention also provides a kit containing the transdifferentiation culture medium A, transdifferentiation culture medium B, and spermatogonial stem cell culture medium described in the present invention.

[0115] In a preferred embodiment of the invention, the kit further contains SSCs cells, which may be naturally isolated or expanded / passaged cells.

[0116] In a preferred embodiment of the invention, the kit further comprises SSC amplification medium. Preferably, it also comprises neuronal differentiation medium, astrocyte differentiation medium, and / or oligodendrocyte differentiation medium.

[0117] If necessary, the kit also contains culture media / reagents for isolating and maintaining cells. Preferably, the kit also includes instructions for use to facilitate application by those skilled in the art in research or clinical practice.

[0118] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Science Press, or according to the manufacturer's recommendations.

[0119] I. Materials and Methods

[0120] 1. RT-PCR and qRT-PCR

[0121] SSCs and iNSCs in the mid-stage of differentiation, differentiated neurons, astrocytes, and oligodendrocytes were collected using 0.05% trypsin digestion solution. The cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. Total RNA was extracted from the cells using TRIzol lysis buffer and reverse transcribed to synthesize cDNA strands, followed by RT-PCR and qRT-PCR detection.

[0122] 2. Identification by immunofluorescence staining

[0123] Discard the culture medium, rinse the cells with PBS buffer, add 200 μL of 4% paraformaldehyde solution to each well, fix for 30 min at room temperature, wash three times with PBST for 5 min each time; add 200 μL of 0.5% Triton X-100 solution to each well, permeabilize at room temperature for 20 min (permeabilization is not necessary for membrane proteins such as MVH), wash three times with PBST for 5 min each time; add 200 μL of 10% goat serum to each well, block in an incubator at 37°C for 15 min, add diluted primary antibody, and incubate overnight at 4°C. The next day, discard the primary antibody, wash three times with PBST for 5 min each time; add diluted secondary antibody, incubate at room temperature in the dark for 1 h, wash three times with PBST for 5 min each time; counterstain cell nuclei with DAPI for 5-10 min, wash three times with PBST for 5 min each time; add mounting medium containing fluorescence quencher, and image.

[0124] 3. Neuronal electrophysiological detection

[0125] 1) Electrode preparation: Ordinary hard silicate glass microelectrodes (VitalSense Scientific Instruments, China) with an outer diameter of 1.50 mm and an inner diameter of 0.89 mm were drawn in four steps using a microelectrode drawing instrument. After filling the electrode with internal liquid, the electrode impedance was 4-6 MΩ.

[0126] 2) Whole-cell patch-clamp recording: Whole-cell patch-clamp recordings were performed at room temperature (20-25℃) using a patch-clamp amplifier. Experimental parameter settings, data acquisition, and stimulation protocol application were all controlled using the sampling software pClamp 10.7 (MD, USA).

[0127] 3) Stimulation and recording:

[0128] Under voltage clamping, with a clamping voltage of -70 mV, depolarization step voltage stimulation of -70 to +70 mV for 30 ms and a step size of 10 mV was applied to record whole-cell currents at different voltages.

[0129] Under current clamping, with a clamping current of 0 pA, depolarization step current stimulation of -20 to +70 pA, duration of 200 ms, and step size of 10 pA was applied, and the action potentials of cells under different current stimulation intensities were recorded.

[0130] 4. Construction of dual-fluorescent lentiviral vectors and their infection of SSCs

[0131] To better track changes in iNSCs in the mouse hippocampus, the inventors cloned the 194 bp Nestin core promoter and the 720 bp eGFP sequence into the Plvx-mcherry-N1 lentiviral vector, thus obtaining the dual-fluorescent lentiviral vector CMV-mcherry-Nestin core promoter-eGFP. The mcherry fluorescence is carried by the plasmid itself and is used to indicate the efficiency of cell infection, while eGFP specifically characterizes Nestin gene expression. After coating the lentiviral vector with lentivirus, SSCs were infected with lentiviral vectors.

[0132] 5. iNSCs stereotactic brain injection

[0133] SSCs infected with dual-fluorescent lentivirus were transdifferentiated, and eGFP-positive cells were collected by flow cytometry sorting. Then, iNSCs were injected into the bilateral hippocampi of mice using a stereotaxic device (50 × 10⁶ cells per side). 4 To assess the differentiation potential of iNSCs into neurons in the brain, a microsyringe was used. The needle was inserted vertically downwards 1-2 mm into the punched area. 1 μL of iNSC suspension or 1 μL of PBS buffer was injected into each of the CA1 regions of the hippocampus at a rate of 0.06-0.1 μL / min. After injection, the needle was left in place for 2 minutes, then slowly withdrawn at a rate of 1 mm / min. Four weeks after injection, paraffin sections of brain tissue were subjected to immunofluorescence staining.

[0134] 6. Immunofluorescence staining of paraffin sections of brain tissue

[0135] Mice were euthanized by cervical dislocation, and the skull was carefully dissected to obtain brain tissue. The trimmed brain tissue was fixed overnight in 4% paraformaldehyde solution at 4°C, followed by gradient dehydration, tissue clearing, and paraffin infiltration. Sections were then prepared using a microtome to a thickness of 10 μm. After antigen retrieval, immunofluorescence staining was performed.

[0136] II. Examples

[0137] Example 1: Isolation and Identification of Spermatogonial Stem Cells (SSCs)

[0138] 1. Isolation and culture of spermatogonial stem cells (SSCs)

[0139] The mice used in this example were all 6-day-old male F1 generation mice produced by mating DBA / 2 male mice with C57BL6 female mice. SSCs were isolated using a two-step enzymatic digestion method, as follows: The abdominal skin of the mouse was cut open with ophthalmic scissors to fully expose the abdominal cavity. The mouse testes were placed in D-Hanks buffer containing 1% penicillin-streptomycin solution (commercially available penicillin-streptomycin mixture containing 5000 units / mL penicillin and 5000 µg / mL streptomycin, added to the culture medium at a 1% volume ratio) using ophthalmic forceps. After washing thoroughly 3-5 times, the tunica albuginea of ​​the testes was carefully removed with forceps to expose the loose testicular tissue. The testicular tissue was minced and transferred to a 15 mL centrifuge tube. After washing 3 times with D-Hanks buffer containing 1% penicillin-streptomycin, the tube was centrifuged at 300 × g for 5 min, and the supernatant was discarded. Add 5 mL of 1 mg / mL type IV collagenase to a centrifuge tube and resuspend the tissue pellet. Digest in a 37°C water bath for 10-15 min with shaking. The specific digestion time should be determined by the absence of large tissue fragments in the testicular tissue. During digestion, gently pipette the pellet every 3-5 min to ensure complete digestion. Centrifuge at 300 × g for 5 min and discard the supernatant. Add 3 mL of 0.05% trypsin digestion solution to a 15 mL centrifuge tube, resuspend the pellet, and incubate in a 37°C water bath for 5 min. After terminating digestion with 6 mL of culture medium containing 10% fetal bovine serum, remove tissue fragments using a 40 μm cell sieve. Collect the cell suspension in a 50 mL centrifuge tube, centrifuge at 300 × g for 5 min, and discard the supernatant. Resuspend the cells in an appropriate amount of D-Hanks buffer and purify SSCs using immunomagnetic bead sorting. Add 50 μL of Anti-Mouse CD90.2 MagneticParticles to the cell suspension, incubate at 4°C for 30 min, and then collect the magnetic beads using a magnetic rack. Finally, the SSCs bound to the magnetic beads were resuspended in SSC medium (MEM-α basal medium, 20 ng / mL GDNF (glialcellline-derived neurotrophic factor), 20 ng / mL EGF, 10 ng / mL bFGF, 10 ng / mL LIF, 1 mM non-essential amino acids, 2 mM L-glutamine, 25 μg / mL bovine insulin, 1× vitamin (commercially purchased 100× vitamin was added to the medium, resulting in a final working concentration of 1×), 100 μg / mL transferrin, 60 μM putrescine, 60 ng / mL progesterone, 0.1 mM β-mercaptoethanol, 1% penicillin-antibody, 10% fetal bovine serum) and seeded onto an STO cell feeder layer (treated with mitomycin C) for short-term culture. The medium was changed at half volume every 2 days, and the cells were passaged every 5-7 days at a passage ratio of 1:2 or 1:3.

[0140] 2. SSCs identification

[0141] The cells obtained above were identified using RT-PCR and immunofluorescence staining.

[0142] The results are as follows Figure 1 RT-PCR and immunofluorescence staining results showed that, in addition to expressing germ cell markers (including...), in vitro cultured SSCs expressed germ cell markers (including...). MVH) In addition, it specifically expresses multiple SSCs markers and their related genes, including PLZF , OCT4 , ETV5 and GFRα1 These genes are not expressed in STO cells, and PLZF and OCT4 have significant co-localization.

[0143] The above results demonstrate that the SSCs isolation method described in this invention can effectively isolate SSCs and achieve short-term in vitro amplification of SSCs.

[0144] Example 2: Direct transdifferentiation of SSCs into neural stem cell-like cells (iNSCs)

[0145] 1. Transdifferentiation of SSCs

[0146] Short-term cultured SSCs were digested with 0.05% trypsin in a 37°C incubator for 3-5 min. Digestion was terminated with culture medium containing 10% fetal bovine serum. The cells were then centrifuged at 300 × g for 5 min, and the supernatant was discarded. After resuspending in SSC culture medium, the cells were seeded in 0.2% gelatin-coated 35 mm culture dishes to remove feeder cells and other testicular somatic cells as much as possible. After differential adhesion for 2 h, the culture dishes were gently shaken, and the supernatant was collected. Cell counts were performed. Subsequent culture was then carried out as follows:

[0147] (1) Seed approximately 10,000 SSCs per well in 24-well culture plates coated with 10 μg / mL cohesionin (containing SSCs medium) and cultured at 37°C.

[0148] (2) After 24 h, the SSCs medium was replaced with transdifferentiation medium A (SSCs medium + 1 μM retinoic acid), and cultured for another 24 h.

[0149] (3) Replace the transdifferentiation medium A with transdifferentiation medium B (DMEM / F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 μg / mL bovine serum albumin, 25 μg / mL bovine insulin, 10 μM SB431542, 2 mM glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin-dextrin antibiotics, 1% fetal bovine serum, 20 ng / mL bFGF) for transdifferentiation. Change the medium every 2 days and supplement the medium with 20 ng / mL bFGF every day.

[0150] 2. iNSCs amplification and passage culture

[0151] When SSCs transdifferentiated to day 6, the transdifferentiation medium B was discarded, and the cells were rinsed with D-Hanks buffer. An appropriate amount of 0.05% trypsin digestion solution was added to the culture plate, and the plates were incubated at 37°C for 5 minutes. After terminating digestion with basal medium containing 10% fetal bovine serum, the cells were collected in 1.5 mL centrifuge tubes, centrifuged at 300 × g for 5 minutes, and the supernatant was discarded.

[0152] After gently resuspending the cells in an appropriate amount of iNSC amplification medium (DMEM / F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 μg / mL bovine serum albumin, 25 μg / mL bovine insulin, 10 μM SB431542, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin-antibody, 20 ng / mL EGF, 20 ng / mL bFGF), seed the cells at a 1:1 ratio in 24-well plates coated with 10 μg / mL adhesion protein and incubate at 37°C, changing the medium every 2 days. When the cell density reaches approximately 85% (about 5-6 days), perform passage culture at a ratio of 1:3-1:4.

[0153] 3. Culture and transdifferentiation results

[0154] Culture and transdifferentiation results as follows Figure 2 .

[0155] like Figure 2 As shown in Figure A, from day 2 of transdifferentiation, cell morphology begins to change significantly. At this time, some cell colonies disperse, and the cell morphology changes from the original round or oval shape to a bipolar shape, with elongated processes. By day 6 of transdifferentiation, almost no cells with a morphology similar to SSCs can be observed, and the vast majority of cells have become spindle-shaped cells.

[0156] like Figure 2As shown in Figure B, qRT-PCR results indicate that the expression of NSCs-related genes undergoes dynamic changes with the extension of transdifferentiation time, and the expression of some genes ( Vimentin , ID2 and Nrcam The expression level of ) showed a gradual upward trend, while other related genes such as Nestin , Blbp , CD133 , Cdh2 and L1cam The expression levels of [unspecified substance] reached their highest level on day 6 of transdifferentiation, and then gradually decreased from day 8 onwards.

[0157] like Figure 2 As shown in C, compared with SSCs, the expression of SSCs markers and related genes decreased significantly from day 2 of transdifferentiation, and by day 6 of transdifferentiation, the expression of these genes was almost undetectable, indicating that SSCs cells basically transdifferentiated into iNSCs.

[0158] like Figure 2 As shown in Figure D, immunofluorescence staining results indicated that cells on day 6 of transdifferentiation expressed Nestin and Pax6, markers of NSCs. Furthermore, the double positivity rate for Nestin and Pax6 reached over 95%.

[0159] Example 3: Identification of iNSCs' Self-Renewal Capability

[0160] In this embodiment, it is determined whether iNSCs derived from SSCs transdifferentiated have proliferative capacity.

[0161] The transdifferentiated iNSCs (on day 6 of transdifferentiation culture) were passaged using iNSC amplification medium at a ratio of 1:3 to 1:4, and immunofluorescence staining was performed. Each passage was performed every 3-5 days, designated P1, P2, P3, and so on.

[0162] The results showed that P1, P5, and P10 generation iNSCs all expressed NSC markers Nestin and Pax6, as well as the cell proliferation marker Ki67, and there was significant co-localization among the three. Figure 3 (AC). This indicates that iNSCs transdifferentiated from SSCs have proliferative activity.

[0163] Among them, the double positivity rate for Nestin and Pax6 has consistently been above 95%. Figure 3 D) There was no significant difference in the double positivity rate among iNSCs of different generations.

[0164] Example 4: Detection of iNSCs Differentiation Potential: Differentiation into Astrocytes

[0165] To further explore the cellular characteristics of iNSCs, this embodiment investigates the potential of the aforementioned prepared iNSCs to differentiate into astrocytes.

[0166] After collecting and counting iNSCs, 10,000 iNSCs / well were seeded in 24-well plates coated with poly-L-lysine and cohesin and cultured at 37°C for 12 h. Then, the iNSC amplification medium was replaced with astrocyte differentiation medium (DMEM / F12 medium, 1 mM non-essential amino acids, 2 mM L-glutamine, 1% penicillin antibiotics, 10% fetal bovine serum). The medium was changed every 2 days until the differentiation was completed after 4 weeks.

[0167] The results showed that after 4 weeks of culture in astrocyte differentiation medium, the morphology of iNSCs was extremely similar to that of astrocytes, with a star-shaped cell body, a large and round nucleus, and many protrusions around the cell body. Figure 4 A).

[0168] RT-PCR results showed that differentiated cells expressed astrocyte marker genes. Gfap and S100β ( Figure 4 B).

[0169] Example 5: Detection of iNSCs Differentiation Potential: Differentiation into Oligodendrocytes

[0170] To further explore the cellular characteristics of iNSCs, this embodiment investigates the potential of the aforementioned prepared iNSCs to differentiate into oligodendrocytes.

[0171] After collecting and counting iNSCs, 10,000 iNSCs / well were seeded in 24-well plates coated with poly-L-lysine and cohesin and cultured at 37°C for 12 h. Then, the iNSC amplification medium was replaced with oligodendrocyte differentiation medium A (DMEM / F12 medium, 0.5% N2, 500 μM IBMX) and cultured at 37°C for 5 days, with the medium being changed every 2 days. Then, oligodendrocyte differentiation medium A was replaced with oligodendrocyte differentiation medium B (DMEM / F12 medium, 0.5% N2, 200 μM ascorbic acid, 30 ng / mL 3,3,5-triiodothyronine (T3)) and differentiation continued for 7 days.

[0172] Twelve days after iNSCs differentiated into oligodendrocytes, the cells at this time had a cell morphology similar to oligodendrocytes. Figure 5 A).

[0173] RT-PCR results showed that differentiated cells expressed Ng2 , Olig2 and Mbp Oligodendrocyte marker genes ( Figure 5 B). This indicates that iNSCs transdifferentiated from SSCs have the potential to differentiate into astrocytes and oligodendrocytes.

[0174] Example 6: Detection of iNSCs Differentiation Potential: Differentiation into Neurons

[0175] To further explore the cellular characteristics of iNSCs, this embodiment investigates the potential of the aforementioned prepared iNSCs to differentiate into neurons.

[0176] After collecting and counting iNSCs, 20,000 iNSCs were seeded in 24-well plates coated with poly-L-lysine and cohesin and cultured at 37°C for 12 h. The iNSC amplification medium was then replaced with neuronal differentiation medium (DMEM / F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 2 mM L-glutamine, 1% penicillin-antibiotic, 500 nM anti-Hesl oligonucleotide chain, 500 μM 3-isobutyl-1-methylxanthine (IBMX)). During week 1 of differentiation, half of the neuronal differentiation medium was replaced every 3 days. During weeks 2 and 3 of differentiation, the medium was completely replaced every 3 days.

[0177] Next, the inventors focused on the potential of iNSCs to differentiate into neurons (especially GABAergic neurons) in in vitro and in vivo environments.

[0178] like Figure 6 As shown in Figure A, after 3 weeks of differentiation, the cells exhibit a neuron-like morphology, with elongated axons and expression of neuronal hallmark genes. NSE , Tuj1 , NeuN and Map2 ( Figure 6 B).

[0179] qRT-PCR results showed that the expression levels of neuronal marker genes significantly increased with prolonged differentiation time. Figure 6 C).

[0180] Notably, using the neuronal differentiation system provided in this experiment, markers of GABAergic neurons could be detected after 3 weeks of differentiation. Gad65 and Gad67 The expression ( Figure 6 B), and its expression level also shows a gradual upward trend. Figure 6 C).

[0181] Simultaneously, immunofluorescence staining results also indicated that, three weeks after iNSCs differentiated into neurons, they expressed (GABAergic) neuronal markers—MAP2, Tuj1, GAD65, and GAD67. Figure 6 D).

[0182] Importantly, the neuronal electrophysiological testing results showed that inward Na ion and outward K ion currents could be detected under voltage clamping, indicating that the neurons differentiated from iNSCs were fully functional and capable of generating action potentials. Figure 6 E).

[0183] The results above demonstrate that iNSCs can differentiate into fully functional neurons in vitro.

[0184] Example 7: Detection of iNSCs Differentiation Potential: Survival in the Brain and Ability to Differentiate into Neurons

[0185] Four weeks after iNSCs were injected into the hippocampus of mice, paraffin sections of brain tissue were stained with immunofluorescence to determine the survival of iNSCs in the brain and their ability to differentiate into neurons.

[0186] A statistical analysis was performed on mcherry-positive cells in paraffin sections of the whole brain, and approximately 15 × 10⁶ cells were traced. 4 One Mcherry-positive cell ( Figure 7 A, 7B), of which eGFP-positive cells accounted for 13.8% of the total mcherry-positive cells ( Figure 7 C), which means that iNSCs derived from SSCs can survive in the brain.

[0187] Further immunofluorescence staining and statistical analysis showed that these surviving mcherry-positive and eGFP-negative cells had differentiated into neurons, particularly GABAergic neurons. Figure 7 D, 7E).

[0188] The results of the above examples demonstrate that by altering the microenvironment in which SSCs reside, efficient direct transdifferentiation of SSCs into iNSCs can be achieved. The obtained iNSCs exhibit similar proliferative activity to NSCs, as well as the potential to differentiate into neurons, astrocytes, and oligodendrocytes.

[0189] 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 present invention. 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 scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for preparing neural stem cell-like cells, comprising: Starting with spermatogonial stem cells, spermatogonial stem cells are directly transdifferentiated into neural stem cell-like cells; The method for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells includes: (1) Spermatogonial stem cells are cultured in a spermatogonial stem cell culture medium, wherein the spermatogonial stem cell culture medium is a basal culture medium, and: (2) The cells cultured in (1) were cultured in transdifferentiation medium A; the transdifferentiation medium A was spermatogonial stem cell culture medium and retinoic acid; the spermatogonial stem cells were seeded in a container pre-coated with adhesion protein; the culture time was 24±12 hours; the amount of retinoic acid was 0.2~5 μM; (3) Cells cultured in (2) are cultured in transdifferentiation medium B for 3–8 days; wherein the transdifferentiation medium B is a mixed medium of complete culture medium and neural culture medium mixed at a volume ratio of 1:5 to 5:1, and: The complete culture medium is DMEM / F12 medium, and the neural culture medium is Neurobasal medium.

2. The method as described in claim 1, characterized in that, (2) During inoculation, 5,000 to 20,000 cells / well were inoculated into a 24-well plate and cultured at 37±2℃.

3. The method as described in claim 1, characterized in that, (2) The incubation time is 24±8 hours; or (3) The culture time is 3.5 to 7 days.

4. The method as described in claim 2, characterized in that, The spermatogonial stem cell culture medium is the basic culture medium and: 。 5. The method as described in claim 1 or 4, characterized in that, The basal culture medium is MEM-α medium.

6. The method as described in claim 2, characterized in that, The transdifferentiation culture medium B is: 。 7. The method as described in claim 1, characterized in that, The method further includes: (4) The cells cultured in (3) are cultured or passaged in an amplification medium; the amplification medium is based on the transdifferentiation medium B, with serum replaced by EGF, and the amount of EGF is 5 to 100 ng / mL.

8. A method for preparing nerve cells, comprising: (a) Transdifferentiating spermatogonial stem cells into neural stem cell-like cells or cells passaged therefrom using the method described in any one of claims 1 to 7; (b) Further induce the differentiation of the neural stem cell-like cells or passaged cells of (a) into neural cells; said neural cells include: neurons, astrocytes, oligodendrocytes.

9. The method as described in claim 8, characterized in that, The nerve cells are neurons, cultured using a neuron differentiation medium; the neuron differentiation medium includes: a mixed medium of complete medium and neuronal medium, and N2, B27, L-glutamine, anti-Hesl oligonucleotide chains, and 3-isobutyl-1-methylxanthine; or The nerve cells are astrocytes, cultured using an astrocyte differentiation medium; the astrocyte differentiation medium includes: complete culture medium, and non-essential amino acids, L-glutamine, and fetal bovine serum; or The nerve cells are oligodendrocytes, cultured using an oligodendrocyte differentiation medium; the oligodendrocyte differentiation medium includes: complete culture medium and N2,3-isobutyl-1-methylxanthine.

10. The application of the method according to any one of claims 1 to 7, for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells; or, for preparing neural cells, said neural cells comprising: Neurons, astrocytes, oligodendrocytes.

11. A kit for the in vitro preparation of neural stem cell-like cells or neural cells differentiated from them, comprising: Spermatogonial stem cell culture medium, which is a basic culture medium and contains GDNF, EGF, bFGF, LIF, non-essential amino acids, L-glutamine, bovine insulin, vitamins, transferrin, putrescine, progesterone, β-mercaptoethanol, and serum; Transdifferentiation medium A; wherein transdifferentiation medium A is spermatogonial stem cell culture medium and retinoic acid; the amount of retinoic acid is 0.2-5 μM; Transdifferentiation medium B; the transdifferentiation medium B is: a mixed medium of complete medium and nerve medium mixed at a volume ratio of 1:5 to 5:1, and N2, B27, serum albumin, insulin, SB431542, L-glutamine, β-mercaptoethanol, serum, and bFGF; 。 12. The kit according to claim 11, characterized in that, The kit also includes an amplification medium; the amplification medium is based on transdifferentiation medium B, with serum replaced by EGF, and the amount of EGF is 5-100 ng / mL.

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