Reprogrammed exosome and application thereof in tissue regeneration medicine

Abstract

The application provides a kind of reprogramming cell exosome and its application in tissue regeneration medicine, belong to biological medicine field;The application is through to peripheral blood CD34+Cell is chemically pretreated, dynamic pulse dosing induction, signal path cooperative interception and burst secretion regulation, successfully capture the metastable cell with nerve tendency.The core innovation lies in the use of BSA complexed Ferrostatin-1 throughout the whole process to maintain membrane homeostasis, solve the ferroptosis stress in reprogramming, and combine with tangential flow flexible purification process.The obtained exosome is high in safety, strong in uniformity, and rich in nerve repair active molecules, which significantly improves the biological titer in peripheral nerve regeneration field.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a reprogrammed cell exosome and its application in tissue regeneration drugs. Background Technology

[0002] With the rapid development of biotechnology and regenerative medicine, functional repair after peripheral nerve injury has become a major challenge in clinical neuroscience. While traditional surgical suturing or autologous nerve transplantation can reconstruct anatomical structures to some extent, satisfactory functional recovery is often difficult to achieve due to limitations such as insufficient donor sources, the risk of immune rejection, and a restricted neural regeneration microenvironment. Against this backdrop, inducing the production of bioactive substances with high repair potential using cell reprogramming technology provides a novel theoretical support and technical approach for solving the problem of peripheral nerve regeneration.

[0003] The advent of cell reprogramming technology has revolutionized the paradigm of life sciences. Its core lies in breaking the directional developmental limitations of somatic cells through specific methods, enabling them to regain pluripotency or the ability to transform across lineages. Early reprogramming primarily relied on viral vector-mediated overexpression of transcription factors. Although this approach caused a significant stir in the academic community, the risks of gene integration and the uncontrollability of the induction process severely hampered its clinical translation. Subsequently, the emergence of all-chemical small molecule-induced reprogramming protocols marked a new stage in this field—one that is more controlled, non-integrating, and highly programmed. Research shows that by precisely regulating specific chemical signaling pathways, deep reconstruction of the cellular epigenetic state can be achieved without altering the gene sequence. This all-chemical reprogramming approach not only improves the safety of induction but also provides standardized operational possibilities for industrial-scale production.

[0004] In the research process of reprogramming, the academic community has gradually discovered that the process of cells transitioning from an initial state to a totipotent state does not simply involve a starting point and an ending point. A series of transient and highly active metastable windows exist in between. These intermediate cells often exhibit unique metabolic characteristics and extremely strong paracrine activity. The extracellular vesicles secreted by these metastable cells, especially exosomes, have been shown to be enriched with large amounts of neurotrophic factors, microRNAs, and proteins that can regulate epigenetics. Compared to terminal stem cells, these derivative secretions produced in the metastable stage have stronger biological activity and repair efficacy against specific tissue damage.

[0005] Peripheral nerve regeneration involves axonal growth, myelin formation, and remodeling of distal neural pathways. Exosomes produced by reprogrammed cells possess excellent biocompatibility and the ability to cross physiological barriers due to their natural lipid bilayer structure. They can inhibit excessive inflammatory responses by modulating the immune microenvironment of the damaged area and directly act on damaged Schwann cells and neurons, promoting axonal extension. Currently, obtaining high-performance, highly active exosomes with neurotrophic repair capabilities using chemical reprogramming technology has become a cutting-edge trend in the development of neuroregeneration drugs. Biologics prepared in this way not only avoid the tumorigenic risks associated with cell transplantation but also enable large-scale production through standardized processes, providing a highly valuable clinical candidate for the precision treatment of peripheral nerve injury. Summary of the Invention

[0006] The first objective of this invention is to provide a method for preparing reprogrammed cell exosomes, comprising the following steps: S1 Initiating Cell Acquisition: CD34+ hematopoietic progenitor cells were sorted from peripheral blood to serve as initiating cells; S2 chemical pretreatment: Before induction, the starting cells were subjected to controlled stress culture using a pretreatment medium containing metabolic remodeling agents, lipid membrane enhancers, neural orientation inducing factors and antioxidants. S3 programmed reprogramming induction: Induction is performed using a dynamic pulse dosing method, wherein the dynamic pulse includes the following three timing phases: Phase 1: Add GSK-3 signaling pathway inhibitors and histone deacetylase inhibitors to initiate chromatin remodeling; Phase Two: Withdraw histone deacetylase inhibitors and add TGF-β receptor inhibitors and adenylate cyclase activators to drive lineage switching; Phase 3: Reduce the concentration of GSK-3 signaling pathway inhibitors and add epigenetic interception factors until the metastable characteristic indicators in the cell population reach the preset threshold. S4 metastable state capture: Switch to interception culture medium containing TGF-β receptor inhibitors, MEK / ERK signaling pathway inhibitors and selective epigenetic regulators for lock culture, so that the cells are stably in a metastable state with a SSEA-4 positivity rate of 5-15%; S5 Explosive Secretion Regulation: Switch to a secretion regulation medium containing extracellular vesicle secretion enhancers and exogenous phospholipid precursors for induction, and collect and purify exosomes; The lipid membrane enhancer is BSA-chelated Ferrostatin-1; and the lipid membrane enhancer is added in all of the steps S2 (chemical pretreatment), S3 (programmed reprogramming induction), S4 (metastable capture), and S5 (burst secretion regulation). The pretreatment medium, dynamic pulse drug delivery, interception medium, and secretion regulation medium are all based on DMEM / F12. The culture conditions for steps S2 (chemical pretreatment), S3 (programmed reprogramming induction), S4 (metastable capture), and S5 (explosive secretion regulation) are 37°C, 3-5% O2, and 5% CO2.

[0007] Preferably, step S1, obtaining the starting cells, specifically involves collecting peripheral blood and screening peripheral blood mononuclear cells; sorting the peripheral blood mononuclear cells to obtain hematopoietic progenitor cells CD34+, thus obtaining the starting cells.

[0008] Preferably, the chemical pretreatment in step S2 specifically includes: Controlled stress culture was performed for 24-48 hours using pretreated medium before formal induction; The pretreatment culture medium consists of the following components added to the basal culture medium: α-Ketoglutaric acid, 0.5-2.0 mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; γ-aminobutyric acid, 10-50 μM; L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; B27, 1×; Insulin-transferrin-selenium, 1×.

[0009] Preferably, step S3, inducing programmatic reprogramming, specifically involves: The starting cells pretreated with S2 were then induced and cultured using a programmed dynamic pulse drug delivery method, which specifically included: From day 1 to day 4, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 1-5 μM; Histone deacetylase inhibitors, 0.5-2.0 mM; B27, 1×; L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; From day 5 to day 8, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 1-5 μM; TGF-β receptor inhibitor, 5-20 μM; Adenylate cyclase activator, 5-50 μM; B27, 1×; L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; On day 9 and thereafter, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 0.25-1.25 μM; TGF-β receptor inhibitor, 5-20 μM; Adenylate cyclase activator, 5-50 μM; Epigenetic interception factors, 0.1-2.0 μM; B27, 1×; L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; When the metastable characteristic index in the cell population reaches the preset threshold, the S3 phase is terminated and the process enters the S4 metastable capture phase.

[0010] Preferably, step S4, metastable state capture, specifically involves: Switch to intercept culture medium for 24-72 hours of locked culture; the intercept culture medium is composed of the basal culture medium supplemented with the following substances. TGF-β receptor inhibitors, 2-10 μM; MEK / ERK signaling pathway inhibitors, 0.5-2.0 μM; Selective epigenetic regulators, 0.1–5.0 μM; B27, 1×; L-Ascorbic acid, 100-500 μM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; HEPES, 10-20mM; Sodium pyruvate, 0.5-2.0 mM; The successfully captured cells exhibited biological characteristics: the positivity rate of the cell surface marker SSEA-4 remained stable at 5-15%.

[0011] Preferably, step S5, burst secretion regulation, specifically comprises: After completing the S4 metastable state capture, the cells were switched to secretion regulation medium for secretion induction, and then the culture supernatant was collected and the exosomes were purified. The secretion regulation culture medium is composed of the following substances added to the basic culture medium. Extracellular vesicle secretion enhancer, 1-100 nM; Exogenous phospholipid precursors, 2.5-25 μM; B27, 1×; L-Ascorbic acid, 200-500 μM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; HEPES, 10-20mM.

[0012] Preferably, a complete medium replacement mode is used when switching culture media.

[0013] Preferably, in step S3, the metastable characteristic index of the cell population is when the negative rate of the initial cell surface marker is ≥95% and the positive rate of the pluripotency-related marker is between 5-20%.

[0014] Preferably, in step S5, burst secretion regulation, the collection and purification of exosomes specifically involves: Collect the supernatant after S5 induction culture, and filter it through a microporous membrane to obtain the filtrate; The filtrate is concentrated by tangential flow filtration with a molecular weight cutoff of 100-300 kDa, and the shear force of the tangential flow filtration is controlled at 1000-3000 s. -1 Exosomes were obtained.

[0015] A second objective of this invention is to provide an exosome prepared by the above-described preparation method.

[0016] A third objective of this invention is to provide the application of the aforementioned prepared exosomes in the preparation of peripheral nerve regeneration drugs.

[0017] The technical logic of this invention is based on the synergistic mechanism of epigenetic flexible remodeling and real-time intervention in cellular lipid membrane homeostasis. In the S2 chemical pretreatment stage, the catalytic activity of intracellular dioxygenases is enhanced by increasing the substrate concentration mediated by α-ketoglutarate, thereby pre-initiating the chromatin demethylation process.

[0018] By utilizing BSA-chelated Ferrostatin-1 in synergy with L-ascorbic acid, a lipid antioxidant defense system of the cytoplasmic membrane and organelle membranes was constructed before the application of induction stress. The cell membrane potential was regulated by γ-aminobutyric acid, which enabled the initiating cells CD34+ to enter a controlled hypermetabolic stress state, accumulating energy substrates and homeostatic reserves for subsequent epigenetic remodeling.

[0019] In the S3 programmed reprogramming induction phase, a dynamic pulsed dosing strategy based on temporal fluctuations was employed. Phase one utilized a GSK-3 inhibitor to activate the Wnt signaling pathway, combined with a histone deacetylase inhibitor (HDACi) to significantly reduce chromatin condensation and relieve transcriptional repression of endogenous lineage-restricting genes. Phase two involved withdrawing HDACi and introducing a TGF-β receptor inhibitor to block differentiation pathway signals, utilizing an adenylate cyclase activator to increase intracellular cAMP levels, driving epigenetic transformation towards pluripotency. Phase three achieved a non-linear slowdown of the reprogramming rate by reducing the GSK-3 inhibitor concentration, and introduced epigenetic interceptors (such as EZH2 inhibitors) to dynamically adjust the distribution of repressive modifications such as H3K27me3. Real-time monitoring of the expression slope of markers such as SSEA-4 precisely identified and captured the optimal kinetic window for cells to enter metastable state.

[0020] The interception mechanism in the S4 stage involves jointly inhibiting the TGF-β and MEK / ERK signaling pathways, blocking the transcriptional drive for further cellular evolution towards a fully pluripotent state. This is combined with selective epigenetic regulators to chemically anchor the current chromatin conformation, causing cellular arrest within a specific epigenetic landscape to form a metastable state with highly efficient exosome synthesis potential.

[0021] This stage utilizes sodium pyruvate to enhance mitochondrial oxidative phosphorylation efficiency, along with Ferrostatin-1 maintained throughout the process to resist oxidative stress caused by severe pathway interception, ensuring that the metastable cell population maintains a 5-15% SSEA-4 positivity rate while cytoplasmic multivesicular bodies (MVBs) accumulate excessively.

[0022] In stage S5, by switching to a secretion-regulated environment, the physical squeezing effect created by low-volume culture, combined with extracellular vesicle secretion enhancers (such as calcium ion carriers or neutral sphingomyelinase activators), instantaneously triggers the fusion mechanism between MVBs and the cell membrane, achieving a burst release of loaded exosomes. During this process, exogenous phospholipid precursors directly participate in repairing cell membrane structural damage caused by secretion, ensuring cell survival during the secretion window. Finally, through tangential flow filtration technology, under specific shear force thresholds, and based on differences in hydrodynamic radius and molecular weight cutoff, the targeted recovery and concentration of high-purity, highly bioactive exosomes are achieved.

[0023] The beneficial effects of this invention are as follows: (1) Safety and controllability of the all-chemically induced system: The use of a small-molecule chemical induction system avoids the risks of exogenous gene integration and immunogenicity associated with traditional biological induction methods (such as viral vectors and transcription factor transfection). The complete chemical components have well-defined molecular weights and pharmacokinetic characteristics, enabling the reprogramming process to have higher reproducibility and accuracy in both time and space dimensions, providing the necessary component validation basis for large-scale standardized preparation of clinical-grade exosomes.

[0024] (2) The chemical pretreatment stage enables the starting cells to achieve steady-state function: By employing a short-term chemical pretreatment in the S2 phase, the activity of epigenetic modifying enzymes was pre-regulated using α-ketoglutarate and neural orientation inducing factors, significantly shortening the lag phase for cells to cross the lineage barrier. This phase, through metabolic remodeling, pre-adapted the CD34+ cells of the initiating cells to hypoxic stress, improving cell survival and metabolic adaptability during subsequent high-intensity reprogramming processes, and reserving cellular energy substrates for high-yield exosome synthesis.

[0025] (3) Precise regulation of reprogramming kinetics by dynamic time-sequential pulse dosing: The dynamic pulsed drug delivery method used in the S3 phase simulates the nonlinear fluctuations of signaling pathways during embryonic development by switching chemical signal combinations in stages. Compared to a constant concentration dosing strategy, pulsed drug delivery effectively avoids cell fatigue and apoptosis caused by long-term suppression of signaling pathways. By precisely controlling the timing of action of factors such as GSK-3 and HDAC, it maximizes the stimulation of cell remodeling potential and significantly improves induction efficiency.

[0026] (4) Metastable and efficient capture based on time-series pulses and chemical interception: By implementing a gradient reduction of GSK-3 signaling pathway inhibitors at the end of the S3 phase, combined with synergistic antagonism of TGF-β and MEK / ERK signaling pathways in the S4 phase, this invention achieves directional braking of transcriptome evolution at the characteristic extreme points of the reprogramming kinetic curve. This truncation mechanism based on dynamic regulation of chemical signals overcomes the limitations of traditional reprogramming processes, where cell populations are highly dynamic and difficult to remain in intermediate states. It ensures that the cell population can be stably locked in the epigenetic metastable state window where multivesicle body (MVB) generation efficiency is highest, guaranteeing the uniformity and controllability of the biological characteristics of exosome products.

[0027] (5) BSA complexation of Ferrostatin-1 maintains membrane homeostasis throughout the process: This invention is the first to introduce BSA-chelated Ferrostatin-1 into the entire preparation process. This component, as a highly effective lipid peroxidation inhibitor, effectively blocks the ferroptosis pathway induced by small-molecule reprogramming drugs. By continuously maintaining membrane homeostasis during stages S2 to S5, it not only significantly reduces the risk of cell disintegration due to oxidative stress, but more importantly, it ensures the biological activity of multivesicular bodies during cell membrane fusion, resulting in secreted exosomes with more intact membrane structures and higher biomass.

[0028] (6) The synergistic effect of the integration of explosive secretion and flexible purification processes: In stage S5, the introduction of exogenous phospholipid precursors and secretion enhancers triggers the targeted excretion of MVBs through a chemical extrusion effect. Combined with shear-force-controlled tangential flow filtration technology, this invention achieves an order-of-magnitude increase in exosome production while maximally preserving the integrity of exosome surface proteins and internal nucleic acids through a flexible physical environment, ensuring the biological efficacy of the final product in the field of neural regeneration.

[0029] (7) Enhancement of neural orientation function of metastable exosomes (RAEs): Because this method anchors cells in a metastable state with neural predisposition, the resulting exosomes are naturally enriched with characteristic molecules that promote axonal growth and synaptic repair in terms of proteomics and nucleic acid profiling. Compared to ordinary stem cell exosomes, the exosomes obtained by this invention exhibit stronger targeting and bioactivity in inducing neuronal regeneration and microenvironment repair. Attached Figure Description

[0030] Figure 1a and Figure 1b The results are from the flow cytometer test in Example 1 of this invention.

[0031] Figure 2 The results are the cell morphology observations of Example 1 of the present invention.

[0032] Figure 3 This is a C11-BODIPY fluorescent staining photograph from Example 2 of the present invention.

[0033] Figure 4 This is a morphological observation of Embodiment 2 of the present invention.

[0034] Figure 5 These are metastable capture microscope images from Embodiment 4 of the present invention. The left side shows group 4a, and the right side shows group 4b.

[0035] Figure 6 The transmission electron microscope (TEM) images of the burst secretion regulation in Example 5 of this invention are group 5a.

[0036] Figure 7This is a schematic diagram of protein biomarker detection (Western Blot) in Example 5 of the present invention. The left side is group 5a; the right side is group 5b.

[0037] Figure 8 These are synaptic growth analysis (immunofluorescence) images of the repair effect of metastable exosomes on damaged neurons in Example 6 of this invention, where A is the control group, B is the damaged group, C is the conventional exosome group, D is the RAE 5a group, and E is the RAE 5b group. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental methods used in the specific implementation methods are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0040] In this invention, unless otherwise specified, "%" represents a percentage by mass; the raw materials and reagents used are all commercially available products.

[0041] The BSA-complexed Ferrostatin-1 used in this invention is prepared using the following method: A 10 mM Ferrostatin-1 stock solution (dissolved in DMSO) was slowly added dropwise to a 5% fatty acid-deficient BSA solution (molar ratio 1:2) under light-protected magnetic stirring. The mixture was incubated at 4°C for 2 hours in the dark to allow Fer-1 to fully embed into the hydrophobic cavities of albumin. After preparation, the solution was filtered through a 0.22 μm filter to obtain the BSA-Fer-1 complex premix. The final working concentration of this premix in the culture medium at each stage was controlled within the range of 0.5–5.0 μM.

[0042] Example 1: Obtaining and identifying CD34+ starting cells 1.1 Peripheral blood sample collection and pretreatment Collect 50 mL of peripheral blood from healthy adult volunteers and place it in a vacuum blood collection tube containing disodium EDTA as an anticoagulant. Mix the collected blood thoroughly with an equal volume of phosphate-buffered saline (DPBS, calcium and magnesium ion-free) under aseptic conditions.

[0043] 1.2 Density gradient centrifugation separation of PBMCs Add 15 mL of Ficoll-Paque lymphocyte separation medium to a 50 mL centrifuge tube. Slowly add the diluted blood to the surface of the separation medium, ensuring a clear interface. Centrifuge at 400 g for 20 minutes at room temperature (20-25℃), setting both acceleration and deceleration to the lowest setting (0). After centrifugation, collect the white, cloudy mononuclear cells (PBMCs) in the middle layer using a pipette and place them in a new centrifuge tube. Wash twice with 3 volumes of DPBS, centrifuging at 300 g for 10 minutes each time, and discard the supernatant.

[0044] 1.3 Forward sorting of CD34+ cells using immunomagnetic beads Screening was performed using a CD34 immunomagnetic bead sorting kit (positive selection method): Cell counting and resuspension: according to every 10 8 Each PBMC cell was resuspended in 300 μL of sorting buffer (PBS containing 0.5% BSA and 2 mM EDTA).

[0045] Antibody incubation: Add 100 μL of FcR blocking agent and 100 μL of CD34 magnetic beads, and incubate at 4°C in the dark for 30 minutes.

[0046] Column sorting: The incubated cell suspension is passed through a sorting column (LS column) placed in a magnetic field. Wash three times with sorting buffer to remove unbound CD34- cells.

[0047] Elution and collection: Remove the sorting column from the magnetic field, add 5 mL of sorting buffer, and wash the CD34+ cells bound to the column into a centrifuge tube.

[0048] 1.4 Cell quality evaluation Purity identification: 1% of sorted cells were added and stained with PE-labeled anti-human CD34 antibody, and then detected by flow cytometry.

[0049] like Figure 1a and Figure 1b As shown, Figure 1a Figure 1A shows the flow cytometry scatter plot for the isotype control group; Figure 1B shows the flow cytometry scatter plot for the sample group.

[0050] The specific antibodies used are as follows: FITCanti-humanLineage(CD3 / 14 / 19 / 20 / 56), Biolegend, 348701.

[0051] APC / Fire™750anti-humanCD38, Biolegend 3, 03545.

[0052] PE / Cyanine7anti-humanCD38, Biolegend, 356608.

[0053] APCanti-humanCD90(Thy1), Biolegend, 328113.

[0054] PerCPanti-humanHLA-DR, Biolegend, 307628.

[0055] The results are as follows: Sample numbers: CD34, CD38, CD90, HLA-DR, Lin- Sample: 98.00%, 0.44%, 99.90%, 99.40%, 0.07% Isotype: 0.83%, 0%, 0.04%, 0.08%, 0.02% Viability testing: Cells were counted using trypan blue staining, and the results showed that the viability of sorted cells was ≥98%.

[0056] Morphological observation: Under an inverted microscope, CD34+ cells appeared round, translucent, with plump cytoplasm, and no obvious deformation or aggregation; as shown in the attached image. Figure 2 As shown.

[0057] Example 2: Chemical pretreatment of starting cells 2.1 Preparation of pretreatment culture medium Using DMEM / F12 as the basal culture medium, each chemical component was added at the following final concentrations, and the mixture was divided into low-value and high-value groups.

[0058] Metabolic remodeling agent: α-ketoglutarate (α-KG), 0.5mM / 2.0mM; Lipid membrane enhancer: BSA-complexed Ferrostatin-1 (Fer-1), 0.5 μM / 5.0 μM; Neural orientation inducing factor: γ-aminobutyric acid (GABA), 10μM / 50μM; Antioxidant and nutritional system: L-ascorbic acid 100μM / 500μM; HEPES, 10mM / 20mM; B27, 1× (both groups are the same); Insulin-transferrin-selenium supplement (ITS), 1× (same for both groups).

[0059] After all components are added, sterilize by filtration through a 0.22μm filter membrane. Prepare fresh before use.

[0060] 2.2 Controlled stress culture operation Cell seeding: CD34+ cells obtained from sorting in Example 1 were seeded at a rate of 5 × 10⁶ cells / year. 4 Cells / cm² were seeded in fibronectin-coated culture dishes.

[0061] Environmental control: The culture dishes were placed in a three-gas incubator, with the following parameters set: 37℃, 4% O2 (with an allowable fluctuation range of ±1%), and 5% CO2. The hypoxic environment, combined with α-KG-induced pseudo-hypoxia, was used to initiate the pre-activation of epigenetic modifying enzymes.

[0062] Culture duration: Continuous culture for 48 hours, during which time cell morphology changes are observed without changing the medium.

[0063] 2.3 Evaluation of Pretreatment Effect Stress resistance assessment: C11-BODIPY staining was performed using the lipid peroxidation probe. Results are as follows: Figure 3 As shown, staining with the lipid peroxidation probe C11-BODIPY was performed. The results are as follows. Figure 3 As shown in the figure, from left to right, the cells are: 2a (low value group), 2b (high value group), and control group (basal culture medium only). Flow cytometry histograms show that under the same hypoxic stress conditions, the fluorescence intensity distribution of cells in groups 2a and 2b was generally lower than that in the control group, and the proportion of the M1 high fluorescence region was significantly lower in both groups, indicating that Fer-1 treatment effectively reduced lipid peroxidation levels.

[0064] Morphological evolution: such as Figure 4 As shown in the figure, 2a (low value group) on the left and 2b (high value group) on the right; it was observed that the CD34+ cells in both groups started to show a slight tendency to adhere to the wall from the original suspension / semi-suspension state, the cell body slightly increased, and the nucleolus became obvious, indicating that they had the physiological basis to enter the high-intensity reprogramming stage.

[0065] Example 3: Induced Programmatic Reprogramming This embodiment follows the parallel induction experiments conducted on groups 2a (low value group) and 2b (high value group) in Embodiment 2.

[0066] 3.1 Parallel experimental group setup Experimental group 3a: Cells pretreated with the same method as in Example 2a were used, and the lower limit of the component concentration was used for induction at each stage of S3.

[0067] Experimental group 3b: Cells pretreated with the same method as group B in Example 2 were used, and the upper limit of the component concentration was used for induction at each stage of S3.

[0068] 3.2 Programmed Dynamic Pulse Drug Delivery Operation Both parallel experiments were conducted with the following timing sequence for changing the culture medium. The basal culture medium was DMEM / F12: 0.5 μM BSA-Fer-1 was added to experimental group 3a; 5.0 μM BSA-Fer-1 was added to experimental group 3b; the culture environment was exactly the same as in Example 2.

[0069] Phase 1 (Days 1-4): Group 3a: CHIR99021 1μM; VPA (HDACi) 0.5mM, B27 1×; L-ascorbic acid 100μM; HEPES 10mM; Group 3b: CHIR99021 5μM; VPA (HDACi) 2.0mM, B27 1×; L-ascorbic acid 500μM; HEPES 20mM.

[0070] Phase Two (Days 5-8): Group 3a: CHIR99021 1μM, 616452 (RepSox) 5μM, Forskolin 5μM; B27 1×; L-ascorbic acid 100μM; HEPES 10mM; Group 3b: CHIR99021 5μM, 616452 (RepSox) 20μM, Forskolin 50μM; B27 1×; L-ascorbic acid 500μM; HEPES 20mM.

[0071] Phase Three (Day 9 and beyond): Group 3a: CHIR99021 0.25μM, 616452 (RepSox) 5μM, Forskolin 5μM; 5-Aza (Decitabine) 0.1μM, B27 1×; L-ascorbic acid 100μM; HEPES 10mM; Group 3b: CHIR99021 1.25μM, 616452 (RepSox) 20μM, Forskolin 50μM; 5-Aza (Decitabine) 2.0μM, B27 1×; L-ascorbic acid 500μM; HEPES 20mM.

[0072] 3.3 Determination of the Induction Endpoint and Evaluation of the Effect Dynamic monitoring: Starting from day 9, cell surface markers were sampled daily by flow cytometry.

[0073] Endpoint data: Group 3a (low concentration): Monitoring showed that the SSEA-4 positivity rate increased stepwise; on day 12, the proportion of CD34- was 95.8% and the proportion of SSEA-4+ was 6.2%, which met the metastable threshold, and S3 was terminated.

[0074] Group 3b (high concentration): Monitoring showed that its progression was significantly faster than that of Group 3a. On day 10, the proportion of CD34- was detected to be 97.2% and the proportion of SSEA-4+ was 14.5%, which met the metastable threshold, and S3 was terminated.

[0075] Example 4 Metastable state capture This embodiment follows the 3a group (low value group) and 3b group (high value group) in Embodiment 3, respectively.

[0076] After completing the S3 stage, the medium was switched to capture medium using a full liquid replacement mode. The base medium for the capture medium was DMEM / F12. Experimental group 4a was supplemented with 0.5 μM BSA-Fer-1, and experimental group 4b was supplemented with 5.0 μM BSA-Fer-1. The culture environment was completely consistent with that in Example 2.

[0077] (1) Group 4a (low value group): Added ingredients: SB431542 (TGF-βi) 2μM; PD0325901 (MEK / ERKi) 0.5μM, RG108 0.1μM, sodium pyruvate 0.5mM; B27 1×, L-ascorbic acid 100μM, HEPES 10mM.

[0078] Lockdown time: 72 hours.

[0079] (2) Group 4b (high value group): Added ingredients: SB431542 10μM, PD0325901 2.0μM, RG108 5.0μM, sodium pyruvate 2.0mM; B27 1×, L-ascorbic acid 500μM, HEPES 20mM.

[0080] Lockout time: 24 hours.

[0081] 4.3 Metastable State Characterization: At the end of the locked culture, the biological characteristics of both groups of cells were assessed. (1) Quantitative detection of state stability (flow cytometry tracking): Procedure: Cells were sampled non-invasively at 12h, 24h, 48h and 72h (for group 4a) after locked culture, and the SSEA-4 positivity rate was detected.

[0082] Judgment criteria: Compare the S3 endpoint value with the values ​​at each time point during the S4 lock-in period.

[0083] Results: The results showed that the SSEA-4 positivity rate in group 4a remained between 5.5% and 6.5% within a 72-hour lockout period; in group 4b, it remained between 13.5% and 14.5% within 24 hours. The SSEA-4 expression levels in both groups did not fluctuate significantly over time (coefficient of variation < 5%), demonstrating the anchoring effect of the chemical interception mechanism on metastable states.

[0084] (2) Evolutionary blocking assessment (exclusionary testing): Target genes for testing: core pluripotency genes OCT4, NANOG, and SOX2.

[0085] Control group: S4 interceptor factor was removed, and iPSC-like cells were induced to day 18 using S3 medium.

[0086] The experimental data are shown in Table 1 below.

[0087] Table 1

[0088] Note: All values ​​in Table 1 are relative expression levels based on the initial CD34+ expression level (set as 1). -△△Ct value.

[0089] (3) Assay for anabolism (detection of metabolites): Procedure: The formation of intracellular multivesicular body (MVB) precursors was observed using an inverted microscope.

[0090] Result: As Figure 5 As shown, under a high-resolution inverted microscope, the granular texture of the cytoplasm of cells in groups 4a (left) and 4b (right) is significantly enhanced. Although the cell clone edges remain loose, the cell body folds increase. The cell membrane system is in a state of high-load vesicle assembly, which meets the physiological prerequisites for explosive secretion.

[0091] Example 5: Explosive Secretion Regulation This embodiment follows up on groups 4a and 4b in embodiment 4, respectively, to perform exosome induction and large-scale purification.

[0092] The basal medium for the secretion regulation medium used in this embodiment was DMEM / F12: 0.5 μM BSA-Fer-1 was added to experimental group 5a; 5.0 μM BSA-Fer-1 was added to experimental group 5b; the culture environment was completely consistent with that in Example 2.

[0093] 5.1 Burst Secretion Induction Procedure Group 5a: Monensin 1 nM, phosphatidylcholine (PC) 2.5 μM, B27 1×, L-ascorbic acid 200 μM, HEPES 10 mM.

[0094] Group 5b: Monensin 100 nM, phosphatidylcholine (PC) 25.0 μM, B27 1×, L-ascorbic acid 500 μM, and HEPES 20 mM were added.

[0095] Collection: Cell culture supernatant was collected from both groups 48 hours after induction of secretion.

[0096] 5.2 Exosome purification process (TFF flexible purification) Pretreatment: The supernatant was filtered through a 0.22 μm microporous membrane to remove cell debris.

[0097] Tangential flow filtration (TFF): Group 5a: Using a polyethersulfone membrane with a molecular weight cutoff of 100 kDa, with the shear force controlled at 1000 s. -1 It is 20 times concentrated.

[0098] Group 5b: Use a polyethersulfone membrane with a molecular weight cutoff of 300 kDa, and control the shear force at 3000 s. -1 Concentrated 50 times.

[0099] Elution: Elute with an equal volume of DPBS containing 5% trehalose to obtain the exosome product.

[0100] 5.3 Product Quality Evaluation and Identification To verify the performance of the "metastable exosomes (RAE)" of this invention, conventional mesenchymal stem cell exosomes (MSC-Exo) were introduced as a control group: (1) Yield evaluation (NTA testing): Group 5a: Yield approximately 3.2 × 10⁻⁶ 10 particles / mL.

[0101] Group 5b: Production is approximately 8.5 × 10⁻⁶ 10 particles / mL.

[0102] Comparison: Results show that the yield of RAE in this invention is significantly higher than that of MSC-Exo (approximately 0.5 × 10⁻⁶). 10 The particle / mL ratio increased by 6-17 times.

[0103] (2) Particle size and morphology identification: Particle size distribution: NTA results show that the main peaks of the products in groups 5a and 5b are both located between 80-120 nm, and the distribution curves are single-peaked, indicating extremely high purity.

[0104] Electron microscopy observation: such as Figure 6 As shown, the product exhibits a typical "disc-shaped" or "cup-shaped" bilayer film structure under transmission electron microscopy (TEM), with intact edges and a full internal load.

[0105] (3) Protein biomarker detection (Western Blot): Equal amounts of products from groups 5a and 5b, along with lysate of the starting cells (CD34+), were subjected to SDS-PAGE electrophoresis and transferred to a membrane.

[0106] The results are as follows Figure 7 As shown, strong specific bands of exosome-specific proteins CD63, CD81 and TSG101 were observed at the corresponding molecular weight positions in experimental groups 5a and 5b. Moreover, the intensity of the bands increased synchronously with the increase of the induction concentration (group 5b).

[0107] Protein level identification confirmed that the product prepared in Example 5 has typical exosome molecular characteristics, and the product purified by this process does not contain cell debris, and its purity meets the requirements for biological applications.

[0108] Example 6: Repairing effect of metastable exosomes on damaged neurons This embodiment uses an in vitro simulated neural ischemia-reperfusion injury model to verify the bioactivity of the metastable exosomes (RAE) prepared in Example 5.

[0109] 6.1 Experimental Model Establishment and Grouping Cell selection: Primary neurons from the rat cortex were used.

[0110] Injury model (OGD / R): Neurons were placed in sugar-free culture medium and cultured in a three-gas incubator (94% N2, 5% CO2, 1% O2) for 4 hours under hypoxic conditions. The medium was then replaced with normal culture medium and cultured under normoxic conditions to simulate reperfusion injury.

[0111] Experimental Groups: Control group: Neurons cultured normally.

[0112] Damage group (Model): After OGD / R treatment, only an equal volume of DPBS was added.

[0113] Conventional exosome group (MSC-Exo): After OGD / R treatment, 50 μg / mL of mesenchymal stem cell exosomes were added.

[0114] RAE experimental groups (5a / 5b): After OGD / R treatment, 50 μg / mL of exosomes from groups 5a and 5b prepared in Example 5 were added respectively.

[0115] 6.2 Detection of neural repair indicators Cell viability assay (CCK-8): assesses the survival rate of cells after damage.

[0116] Synaptic growth analysis (immunofluorescence): Immunofluorescence staining was performed using microtubule-associated protein MAP2, and the average synaptic length of each group of neurons was calculated using ImageJ.

[0117] 6.3 Experimental Results (1) Cell viability (CCK-8): The results showed that the survival rates of neurons in each group exhibited significant gradient differences: Control group: 100% (benchmark); Damage group (Model): 52% ± 4.5%; Conventional exosome group (MSC-Exo): 68% ± 3.2%; RAE5a group (low value group): 79% ± 3.8%; RAE5b group (high value group): 91%±2.8%.

[0118] Experiments have shown that regardless of whether low or high concentrations of RAE are used, its protective efficacy against damaged neurons is significantly better than that of existing conventional MSC exosomes.

[0119] (2) Synaptic regeneration effect: like Figure 8 As shown, Figure 8 A (control group): Dense synaptic distribution, complete network; Figure 8 B (damage group): Large-scale synaptic breakage, with only remnants of cell bodies remaining; Figure 8 C (conventional exosome group): A small number of synapses were observed to sprout, but no effective network was formed; Figure 8 D (RAE 5a group): Synapses regenerate significantly, their length increases markedly, and network connections begin to form; Figure 8 E (RAE 5b group): Synapses show explosive growth, and the neural network morphology is basically restored.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing reprogrammed cell exosomes, characterized in that, Includes the following steps: S1 Initiating Cell Acquisition: CD34+ hematopoietic progenitor cells were sorted from peripheral blood to serve as initiating cells; S2 chemical pretreatment: Before induction, the starting cells were subjected to controlled stress culture using a pretreatment medium containing metabolic remodeling agents, lipid membrane enhancers, neural orientation inducing factors and antioxidants. S3 programmed reprogramming induction: Induction is performed using a dynamic pulse dosing method, wherein the dynamic pulse includes the following three timing phases: Phase 1: Add GSK-3 signaling pathway inhibitors and histone deacetylase inhibitors to initiate chromatin remodeling; Phase Two: Withdraw histone deacetylase inhibitors and add TGF-β receptor inhibitors and adenylate cyclase activators to drive lineage switching; Phase 3: Reduce the concentration of GSK-3 signaling pathway inhibitors and add epigenetic interception factors until the metastable characteristic indicators in the cell population reach the preset threshold. S4 metastable state capture: Switch to interception culture medium containing TGF-β receptor inhibitors, MEK / ERK signaling pathway inhibitors and selective epigenetic regulators for lock culture, so that the cells are stably in a metastable state with a SSEA-4 positivity rate of 5-15%; S5 Explosive Secretion Regulation: Switch to a secretion regulation medium containing extracellular vesicle secretion enhancers and exogenous phospholipid precursors for induction, and collect and purify exosomes; The lipid membrane enhancer is BSA-chelated Ferrostatin-1; and the lipid membrane enhancer is added in all of the steps S2 (chemical pretreatment), S3 (programmed reprogramming induction), S4 (metastable capture), and S5 (burst secretion regulation). The pretreatment medium, dynamic pulse drug delivery, interception medium, and secretion regulation medium are all based on DMEM / F12. The culture conditions for steps S2 (chemical pretreatment), S3 (programmed reprogramming induction), S4 (metastable capture), and S5 (explosive secretion regulation) are 37°C, 3-5% O2, and 5% CO2.

2. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, The specific steps of obtaining the starting cells in step S1 are as follows: peripheral blood is collected and peripheral blood mononuclear cells are screened out; hematopoietic progenitor cells CD34+ are sorted out from the peripheral blood mononuclear cells to obtain the starting cells.

3. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, The chemical pretreatment step S2 specifically involves: Controlled stress culture was performed for 24-48 hours using pretreated medium before formal induction; The pretreatment culture medium consists of the following components added to the basal culture medium: α-Ketoglutaric acid, 0.5-2.0 mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; γ-aminobutyric acid, 10-50 μM; L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; B27，1×； Insulin-transferrin-selenium, 1×.

4. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, The specific steps of step S3, procedural reprogramming induction, are as follows: The starting cells pretreated with S2 were then induced and cultured using a programmed dynamic pulse drug delivery method, which specifically included: From day 1 to day 4, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 1-5 μM; Histone deacetylase inhibitors, 0.5-2.0 mM; B27，1×； L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; From day 5 to day 8, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 1-5 μM; TGF-β receptor inhibitor, 5-20 μM; Adenylate cyclase activator, 5-50 μM; B27，1×； L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; On day 9 and thereafter, add the following components to the basal culture medium: GSK-3 signaling pathway inhibitor, 0.25-1.25 μM; TGF-β receptor inhibitor, 5-20 μM; Adenylate cyclase activator, 5-50 μM; Epigenetic interception factors, 0.1-2.0 μM; B27，1×； L-Ascorbic acid, 100-500 μM; HEPES, 10-20mM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; When the metastable characteristic index in the cell population reaches the preset threshold, the S3 phase is terminated and the process enters the S4 metastable capture phase.

5. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, The metastable state capture step S4 specifically involves: Switch to intercept culture medium for 24-72 hours of locked culture; the intercept culture medium is composed of the basal culture medium supplemented with the following substances. TGF-β receptor inhibitors, 2-10 μM; MEK / ERK signaling pathway inhibitors, 0.5-2.0 μM; Selective epigenetic regulators, 0.1–5.0 μM; B27，1×； L-Ascorbic acid, 100-500 μM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; HEPES, 10-20mM; Sodium pyruvate, 0.5-2.0 mM; The successfully captured cells exhibited biological characteristics: the positivity rate of the cell surface marker SSEA-4 remained stable at 5-15%.

6. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, The specific steps of step S5, burst secretion regulation, are as follows: After completing the S4 metastable state capture, the cells were switched to secretion regulation medium for secretion induction, and then the culture supernatant was collected and the exosomes were purified. The secretion regulation culture medium is composed of the following substances added to the basic culture medium. Extracellular vesicle secretion enhancer, 1-100 nM; Exogenous phospholipid precursors, 2.5-25 μM; B27，1×； L-Ascorbic acid, 200-500 μM; BSA-complexed Ferrostatin-1, 0.5–5.0 μM; HEPES, 10-20mM.

7. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, When switching culture media, a complete media replacement mode is used.

8. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, In step S3, the metastable characteristic index of the cell population is when the negative rate of the initial cell surface marker is ≥95% and the positive rate of the pluripotency-related marker is between 5-20%.

9. The method for preparing reprogrammed cell exosomes according to claim 1, characterized in that, In step S5, the burst secretion regulation, the collection and purification of exosomes specifically involves: Collect the supernatant after S5 induction culture, and filter it through a microporous membrane to obtain the filtrate; The filtrate is concentrated by tangential flow filtration with a molecular weight cutoff of 100-300 kDa, and the shear force of the tangential flow filtration is controlled at 1000-3000 s. -1 Exosomes were obtained.

10. The use of exosomes prepared by any of the preparation methods described in claims 1-9 in the preparation of peripheral nerve regeneration drugs.

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