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WO2024198382A1 - Procédé pour la transdifférenciation directe de cellules souches spermatogoniales en cellules semblables à des cellules souches neurales - Google Patents

Procédé pour la transdifférenciation directe de cellules souches spermatogoniales en cellules semblables à des cellules souches neurales Download PDF

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WO2024198382A1
WO2024198382A1 PCT/CN2023/131361 CN2023131361W WO2024198382A1 WO 2024198382 A1 WO2024198382 A1 WO 2024198382A1 CN 2023131361 W CN2023131361 W CN 2023131361W WO 2024198382 A1 WO2024198382 A1 WO 2024198382A1
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cells
neural
stem cell
transdifferentiation
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吴际
方乾
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Shanghai Jiao Tong University
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Definitions

  • the present invention belongs to the field of cell biology, and more specifically, the present invention relates to a method for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells.
  • Neurological diseases are characterized by high recurrence, high prevalence, and great harm. According to statistics, the prevalence of neurological diseases is about 5% worldwide. Common neurological diseases include Alzheimer's disease, epilepsy, spinal cord injury, astroglioma, and oligodendroglioma. However, the occurrence of these diseases is closely related to the functional loss or abnormality of neurons, astrocytes, or oligodendrocytes.
  • NSCs Neural stem cells
  • These cells play an important role in maintaining the normal function of the body's nervous system.
  • NSCs have broad application prospects in basic research and clinical treatment of neurological diseases, but the limited number of NSCs in the body limits their clinical application. Therefore, the source of cells is one of the key issues to be solved first in accelerating the clinical treatment of NSCs.
  • the ways to obtain NSCs include direct isolation and extraction from original tissues and differentiation of pluripotent stem cells.
  • Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have multidirectional differentiation potential and are an effective way to replace primary cell separation.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Transdifferentiation refers to the process of reprogramming one type of differentiated cell 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 studies have shown that transient expression of pluripotency factors combined with appropriate neural signal input or direct overexpression of NSCs-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 lentivirus, which makes its safety in clinical application uncertain.
  • the purpose of the present invention is to provide a method and application for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells.
  • a method for preparing neural stem cell-like cells comprising: Cells are starting cells that directly transform spermatogonial stem cells into neural stem cell-like cells.
  • the method for directly transdifferentiating spermatogonial stem cells into neural stem cell-like cells comprises:
  • transdifferentiation medium A comprises a spermatogonial stem cell culture medium and retinoic acid
  • the cells cultured in (2) are cultured in a transdifferentiation medium B;
  • the transdifferentiation medium B comprises: a mixed medium of a complete culture medium and a neural culture medium, and N2B27, serum albumin, insulin, SB431542, L-glutamine, ⁇ -mercaptoethanol, serum, and bFGF; and neural stem cell-like cells are obtained.
  • the method further comprises: (4) culturing or subculturing the cells cultured in (3) with an expansion medium; the expansion medium is based on the transdifferentiation medium B, except that the serum is replaced with EGF.
  • spermatogonial stem cells are isolated from testicular tissue using a two-step enzymatic digestion method.
  • spermatogonial stem cells are seeded in a pre-coated container (such as a well plate); preferably, the container is coated with adhesion protein (10 ⁇ 5 ⁇ g/mL, more preferably 10 ⁇ 3 ⁇ g/mL or 10 ⁇ 2 ⁇ g/mL); preferably, 5000 to 20000 cells/well are seeded in a 24-well plate; preferably, the culture is carried out at 37 ⁇ 2°C.
  • the culture time is 24 ⁇ 12 hours; preferably 24 ⁇ 8 hours; more preferably 24 ⁇ 6 hours (such as 24 ⁇ 4 hours, 24 ⁇ 3 hours, 24 ⁇ 2 hours, etc.).
  • the culture time is more than 2 days; preferably 3 to 8 days; more preferably 3.5 to 7 days (such as 4, 5, 6 days).
  • 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, ⁇ -mercaptoethanol, and serum; preferably, it comprises:
  • the basal culture medium is MEM- ⁇ culture medium.
  • the vitamin may be a finished product with a concentration of 100 ⁇ , which is adjusted to a working concentration of 1 ⁇ when used.
  • the transdifferentiation medium B comprises:
  • the complete culture medium and the mixed culture medium of the neural culture medium are 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 culture medium, and the neural culture medium is Neurobasal culture medium.
  • the amount of retinoic acid is 0.2-5 ⁇ M (preferably 0.4-3 ⁇ M, more preferably 0.7-1.5 ⁇ M, such as 1 ⁇ 0.2 ⁇ M).
  • the amount of EGF is 5-100 ng/mL (preferably 8-60 ng/mL, more preferably 12-40, such as 20 ⁇ 5 ng/mL or 20 ⁇ 3 ng/mL).
  • the spermatogonial stem cells obtained by culture are cultured on a STO cell feeder layer for transdifferentiation.
  • 20 ⁇ 5ng/mL preferably 20 ⁇ 3ng/mL 20 ⁇ 2ng/mL
  • 20 ⁇ 5ng/mL preferably 20 ⁇ 3ng/mL 20 ⁇ 2ng/mL
  • the spermatogonial stem cells obtained by culture express MVH, PLZF, OCT4, ETV5, and GFR ⁇ 1.
  • the neural stem cell-like cells obtained by culture express Vimentin, ID2, Nrcam, Nestin, Blbp, CD133, Cdh2, and L1cam, and lowly express or do not express Plzf, Id4, Nanos2, Nanos3, Neurog2, Sholh1, Bcl6b, Etv5, and Oct4.
  • the neural stem cell-like cells obtained by culture are positive for Nestin and Pax6.
  • a method for preparing neural cells comprising:
  • neural cells include: neurons, astrocytes, and oligodendrocytes.
  • the neural cells are neurons, which are cultured using a neuronal differentiation medium; preferably, the neuronal differentiation medium comprises: a mixed medium of a complete medium and a neural culture medium, and N2, B27, L-glutamine, anti-Hesl oligonucleotide chain, and 3-isobutyl-1-methylxanthine.
  • the neural cells are astrocytes, which are cultured using an astrocyte differentiation medium; preferably, the astrocyte differentiation medium comprises: a complete culture medium, and non-essential amino acids, L-glutamine, and fetal bovine serum.
  • the neural cells are oligodendrocytes, which are cultured using an oligodendrocyte differentiation medium; preferably, the oligodendrocytes include: a complete culture medium, and N2,3-isobutyl-1-methylxanthine.
  • the neuronal differentiation medium comprises:
  • the astrocyte differentiation medium comprises:
  • the oligodendrocyte differentiation medium comprises:
  • the astrocytes obtained by culture (highly) express Gfap and S100 ⁇ .
  • the oligodendrocytes obtained by culture (highly) express Ng2, Olig2, Mbp, etc.
  • the cultured neurons express NSE, Tuj1, NeuN, Map2, Gad65, and Gad67.
  • the cultured neurons are positive for MAP2, Tui1, GAD65, and GAD67.
  • the cultured neurons have the ability to generate action potentials.
  • a neural stem cell-like cell which is obtained by directly transdifferentiating spermatogonial stem cells from spermatogonial stem cells as starting cells.
  • the neural stem cell-like cells express neural stem cell markers Nestin and Pax6; more preferably, the double positive rate of Nestin and Pax6 reaches more than 95%.
  • the neural stem-like cells are obtained by the method according to any one of claims 2 to 5.
  • the above methods and applications are in vitro methods and applications (non-therapeutic, non-diagnostic), and the cells include: cell culture; or, the culture is carried out in a container.
  • kits for preparing neural stem cell-like cells or neural cells differentiated therefrom in vitro comprising:
  • Spermatogonial stem cell culture medium preferably, comprising: a basal culture medium, and GDNF, EGF, bFGF, LIF, non-essential amino acids, L-glutamine, bovine insulin, vitamins, transferrin, putrescine, progesterone, ⁇ -mercaptoethanol, and serum;
  • Transdifferentiation medium A comprises spermatogonial stem cell culture medium and retinoic acid;
  • Transdifferentiation medium B comprises: a mixed medium of a complete medium and a neural medium, and N2B27, serum albumin, insulin, SB431542, L-glutamine, ⁇ -mercaptoethanol, serum, and bFGF;
  • the kit further comprises an expansion medium; more preferably, the expansion medium is based on the transdifferentiation medium B, with serum replaced by EGF;
  • the kit further comprises a neuronal differentiation medium; more preferably, the neuronal differentiation medium comprises: a mixed medium of a complete medium and a neural medium, and N2, B27, L-glutamine, anti-Hesl oligonucleotide chain, and 3-isobutyl-1-methylxanthine;
  • the kit further comprises 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;
  • the kit further comprises an oligodendrocyte differentiation medium; more preferably, the oligodendrocytes comprise: a complete culture medium and N2,3-isobutyl-1-methylxanthine.
  • RT-PCR was used to detect the expression of Ng2, Olig2 and Mbp genes in oligodendrocytes derived from iNSCs.
  • Fig. 6 Potential of iNSCs to differentiate into neurons
  • (C) qRT-PCR was used to detect the expression of neuronal markers at 1, 2, and 3 weeks of iNSCs differentiation into neurons.
  • Electrophysiological detection of iNSCs after 3 weeks of differentiation into neurons the left image indicates the neurons subjected to electrophysiological detection, the middle image indicates sodium and potassium ion currents, and the right image indicates action potentials.
  • the inventors After in-depth research, the inventors have revealed a method for efficiently and directly transdifferentiating spermatogonial stem cells (SSCs) into neural stem cell-like cells (iNSCs).
  • SSCs spermatogonial stem cells
  • iNSCs neural stem cell-like cells
  • the iNSCs have proliferation 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 invention provides an overall optimization scheme with high efficiency, can obtain more iNSCs in vitro within a very short period, and can obtain high-purity iNSCs, wherein the double positive rate of Nestin and Pax6 reaches more than 95%, which is beneficial to downstream cell separation and purification.
  • SSCs are a type of adult stem cell that can pass genetic information to the next generation.
  • SSCs can self-renew and differentiate into sperm to maintain male fertility.
  • SSCs will no longer differentiate into sperm according to the strict spermatogenesis process, but will instead transdifferentiate into other types of cells based on the microenvironment in which they are located.
  • Current studies have found that SSCs can be directly transdifferentiated into hepatocytes, kidney tissue cells, and neurons using in vitro culture systems and tissue culture techniques, but there are no reports of SSCs directly transdifferentiating into iNSCs.
  • the inventors have devoted themselves to the study of 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.
  • the method induces differentiation of spermatogonial stem cells in vitro by rationally designing the transdifferentiation stage and adding key factors for inducing differentiation, thereby obtaining functional neural stem cell-like cells.
  • “functional” means that the neural stem-like cells obtained according to the described method have the same or similar functions as expected.
  • the present invention provides a method for preparing neural stem cell-like cells in vitro.
  • the method comprises: (1) culturing spermatogonial stem cells with a spermatogonial stem cell culture medium; (2) culturing the cells cultured in (1) with a transdifferentiation culture medium A; the transdifferentiation culture medium A comprises a spermatogonial stem cell culture medium and retinoic acid; (3) culturing the cells cultured in (2) with a transdifferentiation culture medium B; and obtaining neural stem cell-like cells.
  • the culture medium may be replaced with a fresh one according to the culture conditions.
  • the method is applicable to the transdifferentiation of spermatogonial stem cells 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, gorillas, etc.), livestock (such as cattle, sheep, dogs, pigs, rabbits, etc.).
  • the medium used for cultivation or for induction is a liquid medium (culture broth).
  • the spermatogonial stem cells can be spermatogonial stem cells from the body, or can be spermatogonial stem cells that have been expanded, cultured, passaged, or established.
  • the inventors have established spermatogonial stem cells in previous studies.
  • a method for preparing spermatogonial stem cells includes: using a two-step enzyme (type IV collagenase + pancreatin) digestion method to separate and prepare spermatogonial stem cells from testicular tissue.
  • the transdifferentiation of SSCs to iNSCs is performed by the following specific steps: (1) spermatogonial stem cells are isolated by a two-step enzymatic digestion method and amplified in vitro with SSCs medium; (2) about 10,000 SSCs/well are seeded in a 24-well plate coated with adhesion protein, and after in vitro culture at 37°C for 24 hours, 1 ⁇ M retinoic acid is added to the SSCs medium and cultured for another 24 hours; (3) the medium is replaced with a 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.
  • the entire culture system uses cell growth factors and chemical molecules, and does not introduce exogenous genes involved in reprogramming or transdifferentiation functions (does not change the genome structure), so it can avoid the interference of exogenous genes on the genome stability of the original stem cells and the transplantation safety hazards related to exogenous cells.
  • neural stem cell-like cells can be efficiently obtained, which have the characteristics of double positive Nestin and Pax6.
  • the efficiently obtained neural stem cell-like cells have a high transdifferentiation efficiency, an ideal cell state, and can well support the further differentiation of neural stem cell-like cells into neural cells.
  • the transdifferentiation method of the present invention can obtain high-purity iNSCs in vitro, wherein the double positive rate of Nestin and Pax6 reaches more than 95%.
  • the transdifferentiation method of the present invention is an orderly operated, integrated scheme, which requires a very short time and is very efficient (for example, only 8 days).
  • the transdifferentiation method of the present invention does not need to go through the pluripotency stage (as shown in the results of Figure 2C, the expression level of the pluripotency gene Oct4 gene shows a decreasing trend during the transdifferentiation process), but can achieve direct adherent transdifferentiation culture, with a simple operation process and a high success rate.
  • the neural stem cell-like cells obtained by the present invention have many applications, including further inducing differentiation into various types of neural cells (including neurons, astrocytes, oligodendrocytes, etc.), achieving efficient differentiation and obtaining neural cells with typical characteristics. Furthermore, these neural cells can form brain cell tissues.
  • the neural stem cell-like cells obtained by the present invention can be enriched at the site of injury, differentiated into nerve cells under the local tissue/microenvironment, and repair and supplement the damaged nerve cells.
  • ischemia and hypoxia can cause such injuries
  • the administration of neural stem cell-like cells means enrichment at the site of injury.
  • the neural stem cell-like cells can also stimulate the original neurons and glial cells by producing a variety of neurotrophic factors, thereby promoting the repair of damaged cells.
  • the neural stem cell-like cells can also strengthen the connection between synapses, establish new neural circuits, and reduce oxidative stress in the brain.
  • the neural stem cell-like cells of the present invention can be applied to a variety of central nervous system diseases, including brain and spinal cord injuries, such as but not limited to: cerebral palsy, meningitis or its sequelae, cerebral dysplasia, stroke (cerebral hemorrhage or cerebral infarction) or its sequelae, brain trauma, spinal cord injury, motor neuron disease, amyotrophic lateral sclerosis, brain atrophy, ataxia, Parkinson's disease, epilepsy, multiple system atrophy, senile dementia or vascular dementia, chorea, multiple radiculitis, neurological deafness, facial paralysis, peripheral neuropathy, etc.
  • brain and spinal cord injuries such as but not limited to: cerebral palsy, meningitis or its sequelae, cerebral dysplasia, stroke (cerebral hemorrhage or cerebral infarction) or its sequelae, brain trauma, spinal cord injury, motor neuron disease, amyotrophic lateral sclerosis, brain atrophy, ataxia, Parkinson's
  • the present invention also provides a culture medium for induction culture at each stage, including the SSCs culture medium, transdifferentiation culture medium A, and transdifferentiation culture medium B.
  • the culture medium can be used to culture SSCs cells and to produce.
  • it is an iNSCs expansion culture medium.
  • cytokines or chemical components known in the art that have the same or similar functions as them can also be applied to the present invention.
  • the analogs, proteins with the same functions (such as proteins with the same functions of growth factors) or compounds of the specifically listed components, equivalent compounds, analogs, derivatives and/or their salts, hydrates or precursors that induce the same target can also be used to replace the above-mentioned specific components to achieve the same technical effects.
  • These analogs, proteins with the same functions or compounds should also be included in the present invention.
  • Analogs of compounds include, but are not limited to, isomers and racemates of compounds. Compounds have one or more asymmetric centers.
  • the "precursor of a compound” refers to a compound that can be converted into a compound of any of the above compounds, or a salt or solution composed of a compound of any of the above compounds in the culture medium after being applied or treated by an appropriate method.
  • the culture medium may also be added with ingredients for preventing bacterial contamination of cell culture, such as Gram-positive and Gram-negative bacterial contamination, such as some antibiotics.
  • ingredients for preventing bacterial contamination of cell culture such as Gram-positive and Gram-negative bacterial contamination, such as some antibiotics.
  • dual antibodies are used.
  • the cytokines or chemical components are added to a suitable basal medium/complete medium/neural medium or mixed medium.
  • the basal medium/complete medium can be MEM- ⁇ , DMEM/F12, DMEM, RPMI1640 or an alternative medium with similar nutritional components;
  • the neural medium can 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 basal cell culture medium.
  • the preferred embodiments of the present invention provide of cell culture medium.
  • the present invention also provides a kit, which contains the transdifferentiation medium A, transdifferentiation medium B and spermatogonial stem cell medium described in the present invention.
  • the kit further comprises SSCs cells, which may be naturally isolated or expanded/passaged cells.
  • the kit further comprises an SSCs expansion medium, preferably, a neuron differentiation medium, an astrocyte differentiation medium and/or an oligodendrocyte differentiation medium.
  • an SSCs expansion medium preferably, a neuron differentiation medium, an astrocyte differentiation medium and/or an oligodendrocyte differentiation medium.
  • the kit also contains culture medium/reagents for isolating and maintaining cells.
  • the kit also contains instructions for use, so as to facilitate the use of the kit by those skilled in the art in research or clinical application.
  • RNA in the cells was extracted with TRIzol lysis solution, and reverse transcribed to synthesize cDNA chains, followed by RT-PCR and qRT-PCR detection.
  • Discard the culture medium rinse the cells with PBS buffer, add 200 ⁇ L 4% paraformaldehyde solution to each well, fix at room temperature for 30min, wash 3 times with PBST, 5min each time; add 200 ⁇ L 0.5% Triton X-100 solution to each well, permeabilize the membrane for 20min at room temperature (for membrane proteins such as MVH, permeabilization is not required), wash 3 times with PBST, 5min each time; add 200 ⁇ L 10% goat serum to each well, block in a 37°C incubator for 15min, add diluted primary antibody, and incubate overnight at 4°C.
  • Electrode preparation Ordinary hard silicate glass microelectrodes with an outer diameter of 1.50 mm and an inner diameter of 0.89 mm (VitalSense Scientific Instruments, China) were drawn by a microelectrode drawing instrument in four steps. After liquid, the electrode impedance is 4-6M ⁇ ;
  • the clamping voltage was -70mV, and depolarizing step voltage stimulation of -70 ⁇ +70mV, duration of 30ms, and step of 10mV was given to record the whole cell current under different voltages.
  • the clamp current was 0pA, and depolarizing step current stimulation of -20 to +70pA, duration 200ms, and step amplitude 10pA was given, and the action potential of the cells under different current stimulation intensities was recorded.
  • the inventors cloned the 194bp Nestin core promoter and the 720bp eGFP sequence into the Plvx-mcherry-N1 lentiviral vector, thereby obtaining a dual-fluorescence lentiviral vector -CMV-mcherry-Nestin core promoter-eGFP, in which the mcherry fluorescence is carried by the plasmid itself to indicate the efficiency of infected cells, and eGFP specifically characterizes the expression of the Nestin gene.
  • SSCs were infected with lentivirus.
  • iNSCs were injected into the hippocampus of both sides of the mouse using a brain stereotaxic instrument (50 ⁇ 10 4 iNSCs were injected on each side) to detect their differentiation potential into neurons in the brain.
  • the microinjector was inserted vertically downward 1-2mm at the punched position.
  • 1 ⁇ L of iNSCs cell suspension or 1 ⁇ L of PBS buffer was injected into the CA1 region of the hippocampus on both sides at a rate of 0.06-0.1 ⁇ L/min. After the injection, the needle was retained for 2min, and then the injection needle was slowly withdrawn at a speed of 1mm/min.
  • paraffin sections of the brain tissue were immunofluorescently stained.
  • the skull was carefully peeled off and the brain tissue was obtained.
  • the trimmed brain tissue was fixed in 4% paraformaldehyde solution at 4°C overnight, and then after gradient dehydration, tissue clearing and wax treatment, it was sliced using a slicer with a slice thickness of 10 ⁇ m. After the slice antigen was repaired, immunofluorescence staining was performed.
  • SSCs spermatogonial stem cells
  • SSCs spermatogonial stem cells
  • mice used in the embodiment are all F1 generation 6-day-old male mice produced by mating DBA/2 male mice with C57BL6 female mice.
  • SSCs were separated by a two-step enzymatic digestion method, and the steps are as follows: cut the mouse abdominal skin with ophthalmic scissors to fully expose the abdominal cavity, and place the mouse testicles in D-Hanks buffer containing 1% double antibody (penicillin-streptomycin solution, commercially available penicillin-streptomycin finished product mixture, the solution contains 5000 units/mL penicillin and 5000 ⁇ g/mL streptomycin, added to the culture medium at a volume ratio of 1%) with ophthalmic forceps, and after fully washing 3-5 times, carefully remove the testicular white membrane with forceps to expose the loose testicular tissue.
  • penicillin-streptomycin solution commercially available penicillin-streptomycin finished product mixture, the solution contains 5000 units/mL penicillin and 5000 ⁇ g/mL streptomycin, added to the
  • testicular tissue After the testicular tissue is cut into pieces, it is transferred to a 15mL centrifuge tube, washed 3 times with D-Hanks buffer containing 1% double antibody, centrifuged at 300 ⁇ g for 5min, and the supernatant is discarded. Add 5 mL 1 mg/mL type IV collagenase to the centrifuge tube and resuspend the tissue pellet. Shake and digest in a 37°C water bath for 10-15 minutes. The specific digestion time is based on the absence of large tissue blocks in the testicular tissue. During the digestion process, gently blow the pellet with a pipette every 3-5 minutes to make it fully digested. Centrifuge at 300 ⁇ g for 5 minutes and discard the supernatant.
  • SSCs culture medium MEM- ⁇ basal medium, 20 ng/mL GDNF (glial cell line-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 ⁇ g/mL bovine insulin, 1 ⁇ vitamin (commercially purchased 100 ⁇ vitamin was added to the culture medium, and the final working concentration was 1 ⁇ ), 100 ⁇ g/mL transferrin, 60 ⁇ M putrescine, 60 ng/mL progesterone, 0.1 mM ⁇ -mercaptoethanol, 1% double antibody, 10% fetal bovine serum) and inoculated on the SSCs culture medium (MEM- ⁇ basal medium, 20 ng/mL GDNF (glial cell line-derived neurotrophic factor), 20 ng/mL EGF, 10 ng/mL bFGF, 10 ng/mL LIF, 1 mM non
  • the cells obtained above were identified by RT-PCR and immunofluorescence staining.
  • RT-PCR and immunofluorescence staining results showed that in addition to expressing germ cell markers (including MVH), SSCs cultured in vitro also specifically expressed multiple SSCs markers and their related genes, including PLZF, OCT4, ETV5 and GFR ⁇ 1, which were not expressed in STO cells, and PLZF and OCT4 had obvious co-localization.
  • germ cell markers including MVH
  • SSCs cultured in vitro also specifically expressed multiple SSCs markers and their related genes, including PLZF, OCT4, ETV5 and GFR ⁇ 1, which were not expressed in STO cells, and PLZF and OCT4 had obvious co-localization.
  • Example 2 Direct transdifferentiation of SSCs into neural stem cell-like cells (iNSCs)
  • transdifferentiation medium A with transdifferentiation medium B (DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 ⁇ g/mL bovine serum albumin, 25 ⁇ g/mL bovine insulin, 10 ⁇ M SB431542, 2 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% double antibody, 1% fetal bovine serum, 20 ng/mL bFGF) for transdifferentiation.
  • transdifferentiation medium B DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 ⁇ g/mL bovine serum albumin, 25 ⁇ g/mL bovine insulin, 10 ⁇ M SB431542, 2 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% double antibody, 1% fetal bovine serum, 20 ng/mL bFGF
  • the transdifferentiation medium B When SSCs were transdifferentiated to the 6th day, the transdifferentiation medium B was discarded, the cells were rinsed with D-Hanks buffer, an appropriate amount of 0.05% trypsin digestion solution was added to the culture plate, and incubated in a 37°C incubator for 5 minutes. After terminating the digestion with a basic medium containing 10% fetal bovine serum, the cells were collected in a 1.5 mL centrifuge tube, centrifuged at 300 ⁇ g for 5 minutes, and the supernatant was discarded.
  • iNSCs expansion medium DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 ⁇ g/mL bovine serum albumin, 25 ⁇ g/mL bovine insulin, 10 ⁇ M SB431542, 2mM L-glutamine, 0.1mM ⁇ -mercaptoethanol, 1% double antibody, 20ng/mL EGF, 20ng/mL bFGF
  • iNSCs expansion medium DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 25 ⁇ g/mL bovine serum albumin, 25 ⁇ g/mL bovine insulin, 10 ⁇ M SB431542, 2mM L-glutamine, 0.1mM ⁇ -mercaptoethanol, 1% double antibody, 20ng/mL EGF, 20ng/mL bFGF
  • cell morphology began to change significantly from the second day of transdifferentiation. At this time, some cell colonies dispersed, and the cell morphology changed from the original round or oval to a bipolar morphology with elongated processes. By the sixth day of transdifferentiation, cells with morphology similar to SSCs were almost not observed, and most cells became spindle cells.
  • the qRT-PCR results showed that with the extension of transdifferentiation time, the expression of NSCs-related genes showed a dynamic change process, and the expression levels of some genes (Vimentin, ID2 and Nrcam) showed a gradual upward trend.
  • the expression levels of other related genes such as Nestin, Blbp, CD133, Cdh2 and L1cam reached the highest level on the 6th day of transdifferentiation, and then gradually decreased from the 8th day.
  • iNSCs obtained by transdifferentiation were subcultured using iNSCs expansion medium at a subculture ratio of 1:3-1:4, and immunofluorescence staining was performed.
  • One generation was performed every 3-5 days, and was counted as P1, P2, P3, and so on.
  • iNSCs were collected and counted, 10,000 iNSCs cells/well were seeded in a 24-well plate coated with poly-lysine and adhesion protein and cultured in a 37°C incubator for 12 h. Then, the iNSCs expansion medium was replaced with astrocyte differentiation medium (DMEM/F12 medium, 1 mM non-essential amino acids, 2 mM L-glutamine, 1% double antibody, 10% fetal bovine serum). The medium was replaced every 2 days until the differentiation was completed after 4 weeks.
  • astrocyte differentiation medium fetal bovine serum
  • RT-PCR results showed that the differentiated cells expressed astrocyte marker genes Gfap and S100 ⁇ ( Figure 4B ).
  • Example 5 Detection of iNSCs differentiation potential: Differentiation into oligodendrocytes
  • iNSCs were collected and counted, 10,000 iNSCs cells/well were seeded in a 24-well plate coated with poly-lysine and adhesion protein and cultured in a 37°C incubator for 12 h.
  • the iNSCs expansion medium was then replaced with oligodendrocyte differentiation medium A (DMEM/F12 medium, 0.5% N2, 500 ⁇ M IBMX) and cultured in a 37°C incubator for 5 days.
  • oligodendrocyte differentiation medium A DMEM/F12 medium, 0.5% N2, 500 ⁇ M IBMX
  • oligodendrocyte differentiation medium A was replaced with oligodendrocyte differentiation medium B (DMEM/F12 medium, 0.5% N2, 200 ⁇ M ascorbic acid, 30 ng/mL 3,3,5-triiodothyronine (T3)) and differentiation continued for 7 days.
  • oligodendrocyte differentiation medium B DMEM/F12 medium, 0.5% N2, 200 ⁇ M ascorbic acid, 30 ng/mL 3,3,5-triiodothyronine (T3)
  • the cells After iNSCs were differentiated into oligodendrocytes for 12 days, the cells had a cell morphology similar to that of oligodendrocytes ( FIG. 5A ).
  • RT-PCR results showed that the differentiated cells expressed oligodendrocyte marker genes such as Ng2, Olig2 and Mbp (Figure 5B), indicating that iNSCs derived from SSCs have the potential to differentiate into astrocytes and oligodendrocytes.
  • oligodendrocyte marker genes such as Ng2, Olig2 and Mbp
  • iNSCs were collected and counted, 20,000 iNSCs were seeded in a 24-well plate coated with poly-lysine and adhesion protein.
  • the iNSCs expansion medium was replaced with a neuronal differentiation medium (DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 2mM L-glutamine, 1% double antibody, 500nM anti-Hesl oligonucleotide chain, 500 ⁇ M 3-isobutyl-1-methylxanthine (IBMX)).
  • DMEM/F12 medium and Neurobasal medium 1:1, 0.5% N2, 1% B27, 2mM L-glutamine, 1% double antibody, 500nM anti-Hesl oligonucleotide chain, 500 ⁇ M 3-isobutyl-1-methylxanthine (IBMX) In the first week of differentiation, the neuronal differentiation medium was replaced by half every 3 days. During the second and third weeks of differentiation, the medium was completely replaced every
  • the inventors focused on the potential of iNSCs to differentiate into neurons (especially GABAergic neurons) in vitro and in vivo.
  • the cells showed a neuron-like morphology, with elongated axons and expressed neuronal marker genes NSE, Tuj1, NeuN and Map2 ( FIG6B ).
  • Example 7 Detection of iNSCs differentiation potential: survival in the brain and ability to differentiate into neurons
  • the results of the above examples show that by changing the microenvironment of SSCs, efficient direct transdifferentiation of SSCs to iNSCs can be achieved.
  • the obtained iNSCs have similar proliferation activity to NSCs, and the potential to differentiate into neurons, astrocytes and oligodendrocytes.

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Abstract

L'invention concerne un procédé pour la transdifférenciation directe de cellules souches spermatogoniales (SSC) en cellules de type cellules souches neurales (iNSC). Les iNSC présentent une activité proliférative, peuvent être cultivées de façon stable in vitro et ont le potentiel de se différencier en d'autres cellules nerveuses, telles que les neurones, les astrocytes et les oligodendrocytes. Le procédé présente une grande efficacité, c'est-à-dire qu'il est possible d'obtenir davantage d'iNSC in vitro dans un délai relativement court, et d'obtenir des iNSC de grande pureté, le taux de double positivité de la Nestine et de la Pax6 étant supérieur ou égal à 95 %.
PCT/CN2023/131361 2023-03-29 2023-11-13 Procédé pour la transdifférenciation directe de cellules souches spermatogoniales en cellules semblables à des cellules souches neurales Pending WO2024198382A1 (fr)

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