Method for electrically stimulating human umbilical cord mesenchymal stem cells in vitro and application

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for electrically stimulating human umbilical cord mesenchymal stem cells in vitro and application. According to the application, the human umbilical cord mesenchymal stem cells are electrically stimulated, and the electrically stimulated human umbilical cord mesenchymal stem cells and the secreted supernatant thereof are collected for detection or in-vitro experiments. The conditions of the electrical stimulation comprise that the current is 5 [mu] A-4 mA, the electric field intensity is 0.625 mV / mm-500 mV / mm, and the frequency is 20 Hz. By adopting the method provided by the invention, the survival, paracrine and differentiation effects of the hUMSCs can be improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method and application of in vitro electrical stimulation of human umbilical cord mesenchymal stem cells. Background Technology

[0002] Spinal cord injury (SCI) is a highly disabling central nervous system injury, with traumatic SCI being the most common clinical manifestation. Despite advancements in modern medicine that have reduced the mortality rate in the acute phase of SCI, its clinical treatment and rehabilitation remain a major challenge for the medical community. SCI leads to neuronal damage, apoptosis, and loss, disrupting ascending and descending neural pathways / circuits and causing persistent impairment of motor, sensory, and autonomic nervous functions. It is well known that the regenerative capacity of the central nervous system is low in adults, and the complex microenvironment of the spinal cord injury site (such as the loss of numerous neurons, immune-inflammatory cascade reactions, cavity formation, and glial scarring) is unfavorable for neuronal survival and axonal regeneration (References 1-2).

[0003] Therefore, nerve regeneration and reconstruction of neural circuit structure and function after spinal cord injury remain key scientific problems that have not yet been solved in the medical field, and are also a hot topic and main focus of international competition in the biomedical field.

[0004] Stem cell therapy for spinal cord injury is one of the more promising methods, and extensive research has been conducted on animal models and clinical trials of stem cell therapy for spinal cord injury. Among various stem cell types, human umbilical-cord mesenchymal stem cell (hUMSC) transplantation may be one of the most attractive treatment options for repairing spinal cord injury. hUMSCs have advantages such as convenient sourcing, easy transportation, low immunogenicity, no ethical controversy, no tumorigenicity, rapid proliferation, and multi-lineage differentiation, and are currently frequently used in basic and clinical research. Studies have shown that hUMSC infusion therapy in patients with spinal cord injury has certain clinical efficacy in relieving sensory and motor disorders, spasticity, and improving bowel and bladder function (Reference 3). However, the survival time of hUMSCs after transplantation or intrathecal infusion in the transplantation site or intrathecal space is insufficient, resulting in unsatisfactory effects in repairing spinal cord injury.

[0005] Document 1: Ahuja CS, Nori S, Tetreault L, Wilson J, Kwon B, Harrop J, ChoiD, Fehlings MG. Traumatic spinal cord injury. Nat Rev Dis Primers, 2017. 3:17018. Document 2: Varadarajan SG, Hunyara JL, Hamilton NR, Kolodkin AL, HubermanAD. Central nervous system regeneration. Cell, 2022. 185(1): 77-94. Document 3: Albu S, Kumru H, Coll R, et al. Clinical effects of intrathecaladministration of expanded Wharton jelly mesenchymal stromal cells inpatients with chronic complete spinal cord injury: a randomized controlled study. Cytotherapy, 2021, 23(2):146-156. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a method and application for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells. This application constructs an in vitro electrical stimulation device and develops an in vitro experimental method for studying the effects of electrical stimulation on improving the survival, paracrine function, and differentiation of hUMSCs.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells, wherein human umbilical cord mesenchymal stem cells are electrically stimulated, and the human umbilical cord mesenchymal stem cells and their secretory supernatant are collected after electrical stimulation for detection or in vitro experiments. The conditions for the electrical stimulation include: a current of 5 μA to 4 mA, an electric field strength of 0.625 mV / mm to 500 mV / mm, and a frequency of 20 Hz.

[0008] The above-mentioned electrical stimulation conditions can effectively reduce hUMSCs apoptosis and promote cell survival, as well as promote hUMSCs proliferation and improve hUMSCs cell viability.

[0009] Furthermore, external electrical stimulation can enhance the secretory function of hUMSCs, especially by promoting the generation of more mitochondria by hUMSCs and their secretion into the extracellular culture medium. This leads to increased cell viability and neurite growth in scratch-damaged spinal cord neurons.

[0010] In addition, electrical stimulation can stimulate the differentiation potential of hUMSCs in the neural direction, and in vitro electrical stimulation can promote the differentiation of hUMSCs into neurons and oligodendrocytes.

[0011] As a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, the conditions for electrical stimulation include: a current of 5 μA to 50 μA, an electric field strength of 0.625 mV / mm to 6.25 mV / mm, and a frequency of 20 Hz.

[0012] Experiments have shown that 5-50 μA in vitro electrical stimulation for 5 days promotes cell survival by reducing apoptosis of hUMSCs, with 50 μA electrical stimulation showing the best effect. 50 μA electrical stimulation for 3 days can also promote the proliferation of hUMSCs.

[0013] 50 μA in vitro electrical stimulation for 3 days can improve the viability of hUMSCs cells, as evidenced by the increased membrane potential of hUMSCs mitochondria after electrical stimulation, and the promotion of hUMSCs to synthesize more TOM20-positive rod mitochondria, forming a rich mitochondrial network in the cytoplasm.

[0014] 50 μA in vitro electrical stimulation can enhance the secretory function of hUMSCs, especially by promoting the generation of more mitochondria in hUMSCs and their secretion into the extracellular culture medium. The uptake of scratch-damaged spinal cord neurons enhances their cell viability and neurite growth.

[0015] Furthermore, 50 μA electrical stimulation can regulate the expression of 2373 significantly differentially expressed genes in hUMSCs, of which 1262 significantly differentially expressed genes were upregulated (such as FGF7, SOD2, TGFβ, FGFR2, ICAM5, laminin, etc.). These significantly upregulated genes are mainly related to mitochondrial biosynthesis and energy metabolism quality control, anti-inflammatory and antioxidant stress, cell adhesion / migration / secretion, angiogenesis and nerve growth.

[0016] Electrical stimulation can activate the differentiation potential of hUMSCs in the neural direction. 1 mA of external electrical stimulation can promote the differentiation of hUMSCs into neurons and oligodendrocytes. EA can synergistically enhance the therapeutic effect of intrathecal infusion of hUMSCs. That is, intrathecal infusion of hUMSCs combined with EA treatment synergistically improves the microenvironment of the spinal cord injury area, promotes the survival of spinal cord neurons and their axonal growth, especially the regeneration of descending nerve fibers or the sprouting of collateral branches that cross the injury area to re-innervate lumbar spinal cord motor neurons, thus promoting the recovery of motor function in the paralyzed hind limbs of beagle dogs.

[0017] As a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, the conditions for electrical stimulation further include a duty cycle DR=50%, a single electrical stimulation time of 30 min, and a continuous electrical stimulation time of 3~5 days.

[0018] In a preferred embodiment of the method for external electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, the electrical stimulation is performed using an external electrical stimulation device; the external electrical stimulation device and the waveform generator are directly connected. The external electrical stimulation device includes a conductive cell culture dish at the bottom and a culture dish lid with a conductive metal sheet at the top. The materials used to prepare the conductive cell culture dish at the bottom include ITO transparent conductive glass, quartz glass rings, and PDMS adhesive. The materials used to prepare the petri dish lid with a conductive metal sheet on top include acrylic, stainless steel, and ABS resin.

[0019] This application utilizes an external electrical stimulation device to design electrical stimulation parameters to enhance the survival, paracrine, and differentiation efficacy of mesenchymal stem cells (MSCs). The external electrical stimulation device is connected to a waveform generator, enabling it to produce low-frequency, continuous-wave alternating current applied to cultured MSCs. The aim is to discover optimal electrical stimulation parameters to improve the survival, paracrine, and differentiation capabilities of MSCs into nerve cells. Furthermore, the paracrine secretions can promote the survival of damaged spinal cord neurons and the growth of their neurites, exerting a neuroprotective function. This will provide a highly efficient source of MSCs and their derived extracellular vesicles for the clinical treatment of patients with spinal cord injuries or other nervous system injuries. It also provides a convenient and feasible in vitro experimental method for exploring the mechanisms of functional electrical stimulation combined with stem cell therapy for spinal cord injuries.

[0020] In a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, in the conductive cell culture dish at the bottom, one side of a quartz glass ring is attached to the center of an ITO transparent conductive glass, the quartz glass ring and the ITO transparent conductive glass are provided with PDMS adhesive, and the ITO transparent conductive glass is connected to the positive / negative electrodes of a waveform generator. And / or, The petri dish lid with a conductive metal sheet on top is assembled from a 3D-printed acrylic transparent lid and a stainless steel electrode using ABS resin fixing posts.

[0021] In a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, in the culture dish lid with a conductive metal sheet on top, the cell culture medium contact surface of the stainless steel electrode is gold-plated using a sputtering gold process, and the other side is welded with a stainless steel cylinder for connecting the negative / positive terminals of the waveform generator.

[0022] In a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, the cell density of the human umbilical cord mesenchymal stem cells is 70%~80%.

[0023] In a preferred embodiment of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells described in this application, the number of human umbilical cord mesenchymal stem cells is 1~5×10⁻⁶. 5 Preferably, the number of human umbilical cord mesenchymal stem cells is 1×10⁻⁶. 5 .

[0024] This application also provides the application of the above-mentioned method of in vitro electrical stimulation of human umbilical cord mesenchymal stem cells in improving the survival, secretory function and neural differentiation potential of human umbilical cord mesenchymal stem cells.

[0025] This application also provides the application of the above-mentioned method of in vitro electrical stimulation of human umbilical cord mesenchymal stem cells in the treatment of spinal cord injury.

[0026] This application describes a method for repairing spinal cord injury using functional electrical stimulation combined with human umbilical cord mesenchymal stem cells. In vitro electrical stimulation of human umbilical cord mesenchymal stem cells can improve their survival rate, paracrine function, and ability to differentiate into nerve cells.

[0027] Compared with the prior art, this application has the following beneficial effects: This application provides a method and application for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells (hUMSCs). The method involves electrically stimulating hUMSCs, collecting the stimulated hUMSCs and their secretory supernatant for testing or in vitro experiments. The electrical stimulation conditions described in this application can reduce hUMSC apoptosis and promote cell survival, as well as promote hUMSC proliferation and cell viability. This is manifested in the increased mitochondrial membrane potential of hUMSCs after electrical stimulation, and the promotion of the synthesis of more TOM20-positive rod mitochondria, forming a rich mitochondrial network in the cytoplasm. In vitro electrical stimulation can enhance the secretory function of hUMSCs, especially by promoting the generation of more mitochondria and their secretion into the extracellular culture medium. This leads to increased uptake by scratch-damaged spinal cord neurons, enhancing their cell viability and neurite growth. Furthermore, electrical stimulation can regulate the expression of 2373 significantly differentially expressed genes in hUMSCs, of which 1262 significantly differentially expressed genes were upregulated (such as FGF7, SOD2, TGFβ, FGFR2, ICAM5, laminin, etc.). These significantly upregulated genes are mainly related to mitochondrial biosynthesis and energy metabolism quality control, anti-inflammatory and antioxidant stress, cell adhesion / migration / secretion, angiogenesis, and nerve growth. Electrical stimulation can stimulate the differentiation potential of hUMSCs in the neural direction, and in vitro electrical stimulation can promote the differentiation of hUMSCs into neurons and oligodendrocytes. EA can synergistically enhance the therapeutic effect of intrathecal infusion of hUMSCs. That is, intrathecal infusion of hUMSCs combined with EA treatment synergistically improves the microenvironment of the spinal cord injury area, promotes the survival of spinal cord neurons and their axonal growth, especially the regeneration of descending nerve fibers or the sprouting of collateral branches that cross the injury area to re-innervate lumbar spinal cord motor neurons, promoting the recovery of motor function in the paralyzed hind limbs of beagle dogs. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of an external electrical stimulation device; Figure 2 This is a cross-sectional diagram of a conductive culture device. Figure 3 Figure showing the results of electrical stimulation promoting the survival and proliferation of hUMSCs; Figure 4 Figure showing the results of 50 μA electrical stimulation enhancing the cell viability of hUMSCs; Figure 5 The results of 50 μA electrical stimulation promoting the secretion of extracellular vesicles, free mitochondria, and mitochondria encapsulated by EVs in hUMSCs are shown in the figure. Figure 6 Figure showing the results of promoting the survival of spinal cord neurons with scratch injury and the growth of neurites in the culture supernatant of hUMSCs after 50μA electrical stimulation; Figure 7The transcriptome sequencing results show that 50 μA electrical stimulation can promote the secretion of more beneficial factors by hUMSCs. Figure 8 The diagram shows the results of 1mA electrical stimulation promoting the differentiation of hUMSCs into neurons and oligodendrocytes. Figure 9 The results of intrathecal infusion of hUMSCs combined with EA significantly reduced glial scarring in the spinal cord injury area of ​​beagle dogs and better promoted nerve fiber regeneration and / or collateral budding into the spinal cord injury area. Figure 10 Imaging evaluations using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) show that intrathecal infusion of hUMSCs combined with EA treatment promotes structural recovery in the spinal cord injury area of ​​a beagle dog. Figure 11 Figure showing the results of intrathecal infusion of hUMSCs combined with EA treatment to promote the recovery of motor function in the paralyzed hind limbs of a beagle. Detailed Implementation

[0029] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0030] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0031] The main instruments, reagents and materials used in this application.

[0032] ITO transparent conductive glass (Shenzhen Micro-Nano Electronics Technology Co., Ltd.), quartz glass ring (Mingyang Quartz Products), acrylic cover (Hefeng Rubber & Plastic Products Factory), 3D printed stainless steel sheet and resin fixing tube (Jialichuang), RIGOL function arbitrary waveform generator (Puyuan), stereo microscope and fluorescence microscope (Leica GmbH, Germany), cryostat (Thermo Fisher), transmission electron microscope (FEI), NanoSight NS300 visual nanoparticle analyzer (Malvern, UK), ultracentrifuge (Beckman), LSM800 confocal fluorescence microscope (Zeiss, Germany), hUMSCs cells (Shanghai Fuyuan Biotechnology), MitoTracker® Red CMXRos (Thermo Fisher), Calcein-AM / PI live / dead cell double staining kit (Solebio Beijing), JC-1 mitochondrial membrane potential detection kit (DOJINDO, Japan), Hoechst 33342 (Sigma), goat serum (GIBCO), primary antibody (Ki67 / TUJ1 / MBP, Abcam), primary antibody (CC1, Oasis) Biofarm secondary antibody (Alex-555 / 488 / 647, Abcam).

[0033] Example 1 This application provides an external electrical stimulation device made of materials such as indium-tin oxide (ITO) transparent conductive film glass and sputtered gold stainless steel sheet, as shown in the schematic diagram. Figures 1-2 As shown.

[0034] The external electrical stimulation device (also known as a conductive cell culture device) consists of a conductive cell culture dish at the bottom and a culture dish lid with a conductive metal sheet at the top.

[0035] Prepare ITO transparent conductive glass and quartz glass ring for the bottom conductive cell culture dish. Prepare PDMS adhesive, coat one side of the quartz glass ring with PDMS and stick it to the center of the ITO transparent conductive glass. Place it in an 80 ℃ oven for 15 min to cure the PDMS and fix it together. Then soak it in ITO glass cleaner for 30 min, rinse it with ultrapure water and dry it.

[0036] The culture dish lid with a conductive metal sheet on top is 3D printed from acrylic (polymethyl methacrylate, usually abbreviated as PMMA), stainless steel sheet, and ABS resin (acrylonitrile-butadine-styrene copolymer, abbreviated as ABS) fixing column. The cell culture medium contact surface of the stainless steel sheet is gold-plated using a sputtering gold process (forming a sputtered gold coating), and the other side is welded with a stainless steel cylinder to connect the negative / positive terminals of the waveform generator. The ABS resin fixing column is fitted onto the stainless steel column, and the transparent acrylic cover passes through the resin fixing column, keeping it parallel to the stainless steel electrode.

[0037] The acrylic transparent cover has a diameter of 4.0 cm, the ITO conductive glass has a diameter of 4.0 cm, and the quartz glass ring has a diameter of 3.5 cm.

[0038] The external electrical stimulation device was sterilized and coated. The conductive cell culture dish at the bottom was directly autoclaved, then coated with laminin or poly-L-lysine (PDL) for 2 h, air-dried in a laminar flow hood for 30 min, washed with PBS for 1 min, and then air-dried for another 30 min before being used for hUMSCs culture. The lid of the culture dish with the conductive metal sheet at the top was soaked twice in 75% alcohol for 30 min each time, then removed and dried in a laminar flow hood, and irradiated with ultraviolet light for 60 min before use.

[0039] Example 2 Human umbilical cord mesenchymal stem cells are electrically stimulated, and the stimulated human umbilical cord mesenchymal stem cells and their secretory supernatant are collected for detection or in vitro experiments.

[0040] This application uses a waveform generator (RIGOL function arbitrary waveform generator) to precisely control parameters such as voltage, frequency, and waveform during electrical stimulation of conductive cell culture dishes. Figure 1 The waveform generator uses a wire to connect one end to the ITO conductive glass part outside the quartz glass ring in the conductive culture device as the positive / negative electrode, and the other end to the top of the stainless steel cylinder as the negative / positive electrode, so as to provide the current for electrical stimulation of the conductive cell culture dish.

[0041] Currently, when treating beagle dogs and rats with electroacupuncture along the Du meridian, the stimulation parameters of the electroacupuncture device are: AC current 1mA, square wave, low frequency 20Hz, etc. The treatment time is 30 minutes per day, with 5 consecutive days of treatment followed by 2 days of rest. The in vivo current (i.e., the recorded output current) during the treatment of beagle dogs and rats with Du meridian electroacupuncture is measured to be in the range of 5~50 μA using an oscilloscope. Based on the in vitro electrical stimulation parameters set for animal electroacupuncture (as shown in Table 1), the current experienced by cells in a monoculture environment is made as consistent as possible.

[0042] The waveform generator parameter settings and experimental groupings are as follows: (1) Control group (no electrical stimulation, all other procedures were performed in the same way).

[0043] (2) Experimental group (different electrical stimulation parameters were set, as shown in Table 1): ① 5 μA ES group: 20 Hz, 30 min, Square Vpp=1.25 mV, high level=1.25 mV, low level=-1.25 mV, duty cycle DR=50%; ②10 μA ES group: 20 Hz, 30 min, Square Vpp=2.5 mV, High level=2.5 mV, Low level=-2.5 mV, Duty cycle DR=50%; ③ 25 μA ES group: 20 Hz, 30 min, Square Vpp=6.5 mV, high level=6.5 mV, low level=-6.5 mV, duty cycle DR=50%; ④ 50 μA ES group: 20 Hz, 30 min, Square Vpp=12.5 mV, high level=12.5 mV, low level=-12.5 mV, duty cycle DR=50%; ⑤ 100 μA ES group: 20 Hz, 30 min, Square Vpp=25 mV, high level=25 mV, low level=-25 mV, duty cycle DR=50%; ⑥ 400 μA ES group: 20 Hz, 30 min, Square Vpp=0.1 V, high level=0.1 V, low level=-0.1 V, duty cycle DR=50%; ⑦ 1 mA ES group: 20 Hz, 30 min, Square Vpp=0.25 V, high level=0.25 V, low level=-0.25 V, duty cycle DR=50%; ⑧2 mA ES group: 20 Hz, 30 min, Square Vpp=0.5 V, high level=0.5 V, low level=-0.5 V, duty cycle DR=50%; ⑨ 4 mA ES group: 20 Hz, 30 min, Square Vpp=1 V, high level=1 V, low level=-1 V, duty cycle DR=50%; ⑩ 6 mA ES group: 20 Hz, 30 min, Square Vpp=1.5 V, high level=1.5 V, low level=-1.5 V, duty cycle DR=50%; ⑪8 mA ES group: 20 Hz, 30 min, Square Vpp=2 V, high level=2 V, low level=-2 V, duty cycle DR=50%.

[0044] Table 1 1. Experiment to verify that external electrical stimulation of hUMSCs improves their survival and secretory function: The following experiments were verified using different groups of external electrical stimulation described above: 1) Calcein-AM / PI live / dead cell staining: After the last electrical stimulation, wait 1.5 h, remove the culture medium from the electroculture wells, and wash twice with 1×Assay Buffer for 2 min each. Add 1-2 μl of Calcein-AM (stock solution) per 1 mL of cells, i.e., add 1 mL of 1×Assay Buffer containing 2 μL Calcein-AM per well, and incubate at 37 ℃ in the dark for 20 min. Add 3 μL of the PI stock solution provided in the kit to the cells and stain at room temperature in the dark for 5 min. After fluorescence incubation, remove the staining solution and wash twice with PBS for 2 min each.

[0045] Note: It is recommended to avoid light throughout the fluorescent staining process. Flow cytometry was used to distinguish between live and dead cell populations and calculate their proportions. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were simultaneously detected under a fluorescence microscope using a 490±10 nm excitation filter. Additionally, a 545 nm emission filter was used to observe only dead cells. Data was collected by observation and photography under a fluorescence microscope.

[0046] 2) JC-1 Mitochondrial Membrane Potential Detection: Add 100 μl of DMSO to a 100 nmol JC-1 Dye tube, and pipette to dissolve the purple-red solid. Store at -20℃. The working solution concentration is 2 μmol / L. Alternatively, transfer 2 μL of 1 mmol / L JC-1 DMSO stock solution to a microtube, add 1 mL of culture medium, and immediately pipette 10 times to mix. Add 1 mL of the prepared JC-1 working solution to each electroculture dish; incubate at 37℃ in a 5% CO2 incubator for 30–60 min. Remove the supernatant and wash the cells twice with HBSS. Observe and photograph under a microscope. When mitochondria are normal and the membrane potential difference remains constant, JC-1 aggregates and emits red fluorescence. When the membrane potential decreases, JC-1 exists as a monomer and emits green fluorescence. Changes in the intensity of red and green fluorescence can be used as indicators of mitochondrial status. Detection conditions: Green: Ex 488 nm / Em 500-550 nm; Red: Ex 561 nm / Em 560-610 nm.

[0047] 3) TOM20 immunofluorescence staining: Cells from one well in each group were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS for 5 min each time, and then incubated with 10% goat serum diluted with 0.3% Triton X-100 at 37°C for 30 min. Samples were incubated overnight with specific primary antibodies (m-TOM20, R-β-actin). The next day, samples were washed three times with PBS for 5 min each time, and then incubated with secondary antibodies at 37°C for 1 h. Cell nuclei were stained with Hoechst 33342 (Hoe) for 10 min. Imaging was performed using a confocal microscope. Image-J Mitochondria Analyzer was used to calculate and statistically analyze mitochondrial aspect ratio, average perimeter, form factor, number of branches, branch lengths, and branch junctions based on hUMSCs Mito-morphology-2D.

[0048] 4) Observe the morphological changes of mitochondria in the culture supernatant after electrical stimulation of hUMSCs using transmission electron microscopy, and track and analyze them using NTA nanoparticles: Collect the supernatant after electrical stimulation, and collect the mitochondria in the supernatant by ultracentrifugation. Ultracentrifugation procedure: Centrifuge the supernatant at 1000 g, 4 ℃ for 5 min to remove cells and cell debris, and centrifuge at 10000 g, 4 ℃ for 60 min to collect the precipitate. Half of the precipitate was fixed with 4% glutaraldehyde at 4℃ for 24 h, gently rinsed twice with PB for 5 min each time, fixed with 1% osmium tetroxide for 1-2 h, gently rinsed twice with PB for 5 min each time, dehydrated in a gradient of 50%, 70%, 80%, 95%, and 100% ethanol for 2-3 min at each gradient, 2-3 min twice with anhydrous acetone, infiltrated with 50% EPON embedding agent (diluted with acetone) for 2 h, infiltrated with 100% EPON embedding agent overnight, and polymerized with 100% EPON 812 at 60℃ for 48 h. A small amount of 100% embedding agent was first added to the template, the precipitate block was placed at the foremost point, and then the embedding agent was added; ultrathin sections were prepared. The other half of the precipitate was resuspended in 1×PBS solution. Then, the instrument and computer software were turned on, and 10 mL of 1×PBS solution was added to the mitochondrial resuspension to obtain a secondary dilution. Before sample testing, the sample cell was rinsed with pure water, and then the sample was injected into the sample cell for observation and particle size analysis data generation.

[0049] 5) Western Blotting: Lyse each group of cells, centrifuge at high speed to remove the precipitate, obtain protein, add 5× loading buffer and ddH2O, centrifuge to mix, heat to 100 ℃ for denaturation for 5 min, centrifuge again, and load the sample. SDS-PAGE electrophoresis, 80V, 90 min; electrophoresis, 250 mA, 240 min. Wash the PVDF membrane with TBST, then block with rapid blocking buffer for 60 min, incubate with primary antibody (m-TOM20, R-GAPDH) at 4 ℃ overnight, wash with TBST 3×10 min, incubate with secondary antibody (goat anti-rabbit / mouse HRP) at room temperature for 60 min, wash with TBST 3×10 min, emulsify with ECL chemiluminescence kit, and expose with a chemiluminescence imaging system.

[0050] 2. Treatment of spinal cord neurons with scratch injury using the supernatant of hUMSCs via electrical stimulation: After electrical stimulation of hUMSCs, their cell culture supernatant was collected and co-cultured with embryonic spinal cord neurons after scratch injury for 48 h. After fixation, TUJ1 immunofluorescence staining was performed, and the number of TUJ1-positive neurons and the average length of neurites in the scratch area were counted and statistically analyzed. Mitotracker Red... +Mitochondria of hUMSCs were labeled using a live-cell mitochondrial staining kit. After electrical stimulation, the cell culture supernatant of hUMSCs was collected and co-cultured with spinal cord neurons after scratch injury for 24 h. After fixation, TUJ1 immunofluorescence staining was performed, and the number of TUJ1 positive neurons and the average length of neurites in the scratch area were counted. After electrical stimulation of hUMSCs, the cell culture supernatant of hUMSCs was collected and co-cultured with lipopolysaccharide-treated embryonic spinal cord neurons for 48 h. After fixation, TUJ1 immunofluorescence staining was performed, and the length of neurites of TUJ1 positive neurons was counted.

[0051] 3. Gene transcriptomics results and validation: 1) Transcriptomics Detection. RNA was extracted and its integrity was accurately detected using an Agilent 2100 bioanalyzer. Libraries were constructed using either the NEB standard library construction method or the strand-specific library construction method (Parkhomchuk et al., 2009). After library construction, preliminary quantification was performed using a Qubit 2.0 Fluorometer, and the library was diluted to 1.5 ng / μL. Subsequently, the insert size of the library was detected using an Agilent 2100 bioanalyzer. Once the insert size met expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (effective concentration higher than 1.5 nM) to ensure library quality. Finally, statistical analysis of gene expression differences was performed, comparing gene expression differences under two or more conditions to identify condition-related specific genes. The biological significance of these specific genes was then further analyzed. The analysis process included quality control, alignment, quantification, differential significance analysis, and functional enrichment.

[0052] 2) qRT-PCR verification of sequencing results. qRT-PCR: Total RNA was extracted from each group of samples using Trizol reagent, and then the total RNA was reverse transcribed into cDNA using a reverse transcription kit. The cDNA was loaded into a 10 μL reaction system according to the instructions of the PCR premixed MIX reagent. The reaction conditions of the CFX96-PCR instrument were set according to the instructions of the real-time PCR kit, and the 10 μL system sample was placed into the PCR instrument for detection.

[0053] The electrical stimulation parameters and functional benefits are shown in Table 2.

[0054] In vitro electrostimulation of hUMSCs with different current parameters revealed that 5–100 μA stimulation for 5 days could reduce apoptosis and promote cell survival to varying degrees, with 50 μA stimulation showing the best effect. Furthermore, 50 μA stimulation for 3 days promoted hUMSC proliferation. When the current intensity was increased to 2 mA, there was no difference in apoptosis rates between the 2 mA and 4 mA stimulation groups and the control group, indicating no adverse effect on hUMSC survival. However, when the current intensity was 6 mA, the apoptosis rate of hUMSCs was higher than that of the control group, affecting hUMSC survival. In vitro electrostimulation of 50 μA for 3 days could improve hUMSC cell viability, manifested in increased mitochondrial membrane potential and promotion of the synthesis of more TOM20-positive rod mitochondria, leading to the formation of a rich mitochondrial network in the cytoplasm. Electrical stimulation of hUMSCs culture supernatants ranging from 5 to 50 μA promoted the survival of spinal cord neurons and the growth of neurites to varying degrees, with 50 μA stimulation showing the best effect. 50 μA stimulation regulated the expression of 2373 significantly differentially expressed genes in hUMSCs, upregulating the expression of 1262 of these genes (e.g., FGF7, SOD2, TGFβ, FGFR2, ICAM5, laminin). These significantly upregulated genes are mainly related to mitochondrial biosynthesis and energy metabolism quality control, anti-inflammatory and antioxidant stress response, cell adhesion / migration / secretion, angiogenesis, and neurogenesis. Furthermore, electrical stimulation can stimulate the neural differentiation potential of hUMSCs; 1 mA in vitro electrical stimulation can promote the differentiation of hUMSCs into neurons and oligodendrocytes. Therefore, 50 μA electrical stimulation is the optimal parameter for promoting hUMSC survival and paracrine function, while 1 mA electrical stimulation is the optimal parameter for stimulating the neural differentiation potential of hUMSCs.

[0055] Table 2 Among them, such as Figure 3 As shown, 5-50 μA in vitro electrical stimulation for 5 days promoted cell survival by reducing hUMSC apoptosis, with 50 μA electrical stimulation showing the best effect. 50 μA electrical stimulation for 3 days can also promote the proliferation of hUMSCs. Figure 3 Figure A: Representative graph showing the results of flow cytometry analysis of PI-labeled dead cells in hUMSCs without electrical stimulation (Control group) and hUMSCs stimulated with 5–100 μA. The dead cell rate was 10.94% in the Control group, 4.74% in the 5 μA group, 5.31% in the 10 μA group, 7.38% in the 25 μA group, 3.47% in the 50 μA group, and 7.82% in the 100 μA group. Figure 3 B: The bar charts show the percentage of dead cells (PI-labeled cells) detected by flow cytometry after electrostimulation of hUMSCs (Control group) and hUMSCs stimulated with 5–100 μA. Compared with the Control group, the percentage of dead cells in hUMSCs stimulated with 5–50 μA ranged from 3% to 8%, with the percentage of dead cells after 50 μA stimulation being only 3.47%. This indicates that electrostimulation with 5–50 μA can reduce apoptosis in hUMSCs, thereby promoting cell survival. p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, n =3. Figure 3 The bar charts in section C show the Calcein-AM / PI live / dead cell staining statistics for hUMSCs without electrical stimulation (Control group) and hUMSCs stimulated with 5–50 μA, respectively. The apoptosis rate was calculated by counting the proportion of dead cells to the total number of cells. Compared with the Control group, the apoptosis rate of hUMSCs stimulated with 5–50 μA was reduced, with the 50 μA group showing the lowest apoptosis rate.* p <0.05,** p <0.01, *** p <0.001, n =3. Figure 3 D: The fluorescent staining diagrams show the staining of live and dead cells of Calcein-AM / PI after electrostimulation of hUMSCs (Control group) and hUMSCs after electrostimulation of 5~50 μA. Green indicates Calcein-AM positive live cells and red indicates PI positive dead cells. Scale bar = 100 μm. Figure 3 E: Fluorescent staining images show Ki67 expression in hUMSCs without electrical stimulation (Control group), and after electrical stimulation of 5 and 50 μA in hUMSCs, respectively. + (purple-red), Hoe (blue), scale bar = 50μm. Figure 3 China F to Figure 3 G: The bar charts show the total cell count and Ki67 of hUMSCs without electrical stimulation (Control group), and hUMSCs after electrical stimulation with 5 and 50 μA, respectively. + Statistical analysis showed that compared with the control group, 5 and 50 μA electrostimulation increased the number of hUMSCs, with the 50 μA group showing the highest cell count. Furthermore, both 5 and 50 μA electrostimulation increased the Ki67 positivity rate of hUMSCs, with the 50 μA group exhibiting the highest Ki67 positivity rate. p <0.001, n =3.

[0056] like Figure 4 As shown, 50 μA in vitro electrical stimulation for 3 days can improve the viability of hUMSCs cells, as evidenced by the increased membrane potential of hUMSCs mitochondria after electrical stimulation, and the promotion of hUMSCs to synthesize more TOM20-positive rod mitochondria, forming a rich mitochondrial network in the cytoplasm. Figure 4 In the middle section, JC-1 staining shows the mitochondrial membrane potential of unstimulated hUMSCs (Control group) and hUMSCs stimulated with 50 μA on days 1, 3, and 5. Red represents JC-1 polymers, green represents JC-1 monomers, and blue represents the cell nucleus Hoe. Scale bar = 50 μm. Figure 4 B: The bar chart shows the statistical differences in mitochondrial membrane potential (calculated JC-1 red / green ratio) on days 1, 3, and 5 after 50 μA electrical stimulation of hUMSCs (Control group) and hUMSCs (Control group). Compared with the Control group, the mitochondrial membrane potential of hUMSCs after 50 μA electrical stimulation was significantly increased on days 1, 3, and 5. p <0.0001, n =3. Figure 4 C: The bar chart shows the changes in cellular ATP content in hUMSCs without electrical stimulation (Control group) and after 50 μA electrical stimulation. Compared with the Control group, the ATP content in hUMSCs significantly increased after 50 μA electrical stimulation. p <0.001, n =3. Figure 4 D: Fluorescent staining of mitochondrial marker Tom20 shows the mitochondrial morphological changes in hUMSCs without electrical stimulation (Control group) and after 50 μA electrical stimulation of hUMSCs. Tom20 (green), β-actin (purple), Hoe (blue). The black and white image is a 2D model generated by ImageJ Mitochondria Analyzer, scale bar = 20 μm. Figure 4 China E to Figure 4 The bar chart shows the statistical analysis of mitochondrial morphology in hUMSCs without electrical stimulation (Control group) and after 50 μA electrical stimulation. The mitochondrial morphology of hUMSCs was analyzed using ImageJ Mitochondria Analyzer. Figure 4 Aspect ratio (length and width) of DTom20 staining Figure 4 (E), Average perimeter ( Figure 4 (F), Form factor (external factors) Figure 4(G), number of branches in the network (G), Figure 4 H), Branch lengths ( Figure 4 (I) Branch junction connection ( Figure 4 Analysis and statistical analysis were performed on hUMSCs (J group). Compared with the control group, hUMSCs showed longer mitochondria and more branches after 50 μA electrical stimulation. p <0.05,** p <0.01, n =8.

[0057] like Figures 5-6 As shown, 50 μA in vitro electrical stimulation can enhance the secretory function of hUMSCs, especially by promoting the generation of more mitochondria by hUMSCs and their secretion into the extracellular culture medium. Figure 5 The uptake of spinal cord neurons damaged by scratches enhances their cell viability and neurite growth. Figure 6 ). Figure 5 China A Figure 5 Image B: Transmission electron microscopy showing free mitochondria in the cell culture supernatant of hUMSCs without electrical stimulation (Control group) and hUMSCs after 50 μA electrical stimulation (indicated by red arrows), scale bar = 2 μm. Figure 5 C, Figure 5 D: Transmission electron microscopy shows mitochondria in extracellular vesicles in cell culture supernatant of hUMSCs without electrical stimulation (Control group) and hUMSCs after 50 μA electrical stimulation (indicated by red arrows), scale bar = 1 μm. Figure 5 E, Figure 5 F:NTA particle size analysis showed extracellular vesicles / mitochondria of different diameter ranges in the cell culture supernatant of unstimulated hUMSCs (Control group) and hUMSCs after 50 μA electrostimulation. Figure 5 G: Western blot results showed Tom20 protein expression in the cell culture supernatant of unstimulated hUMSCs (Control group) and hUMSCs after 50 μA electrostimulation. Figure 5 H: Bar chart showing the gray values ​​and statistical analysis results of Tom20 protein bands in the cell culture supernatant of unstimulated hUMSCs (Control group) and hUMSCs after 50 μA electrostimulation in Western blot. Compared with the Control group, Tom20 protein expression in the cell culture supernatant was significantly increased after 50 μA electrostimulation of hUMSCs. p <0.001, n =3. Figure 5Middle I: Bar chart showing the changes in mitochondrial ATP content in cell supernatant of unstimulated hUMSCs (Control group) and hUMSCs after 50 μA electrostimulation. Compared with the Control group, the mitochondrial ATP content in cell supernatant of hUMSCs significantly increased after 50 μA electrostimulation. *** p <0.001, n =3.

[0058] Figure 6 A: Fluorescent staining illustration shows the uptake and expression of TUJ1 and Mitotracker by TUJ1-positive cells after treating the culture supernatant of Mitotracker-labeled hUMSCs cells with different parameters of electrical stimulation on scratch-damaged neurons. TUJ1 (green), Mitotracker (red), +PBS group is red negative control, arrows indicate TUJ1-positive cell bodies, arrows indicate TUJ1-positive cell processes at the scratch site, scale bar = 25μm. Figure 6 B: The bar chart shows the statistical count of TUJ1 and Mitotracker co-labeled neurons after treating the culture supernatant of Mitotracker-labeled hUMSCs with different electrical stimulation parameters. Compared with the control group, after treating the neurons with the cell supernatant of hUMSCs with 50 μA electrical stimulation, the total number of TUJ1-positive neurons and Mitotracker co-labeled neurons in the scratch injury area was significantly increased. p <0.05,** p <0.01, *** p <0.001, n =3. Figure 6 C: Bar chart shows the statistical distribution of TUJ1-positive neuronal neurite length after treating scratch-damaged neurons with the culture supernatant of Mitotracker-labeled hUMSCs under different electrical stimulation parameters. Compared with the control group, the neurite length of TUJ1-positive neurons in the scratch-damaged area significantly increased after 50 μA electrical stimulation of hUMSCs and treatment with the cell supernatant. p <0.05,** p <0.01, *** p <0.001, n =3.

[0059] like Figure 7As shown, 50 μA electrical stimulation can regulate the expression of 2373 significantly differentially expressed genes in hUMSCs, of which 1262 significantly differentially expressed genes were upregulated (such as FGF7, SOD2, TGFβ, FGFR2, ICAM5, laminin, etc.). These significantly upregulated genes are mainly related to mitochondrial biosynthesis and energy metabolism quality control, anti-inflammatory and antioxidant stress, cell adhesion / migration / secretion, angiogenesis and nerve growth. Figure 7 A: Volcano scatter plot of differentially expressed genes from hUMSCs transcriptome sequencing between the control group (no electrical stimulation) and the 50 μA ES group (50 μA electrical stimulation). Green scatter plots indicate 1111 significantly downregulated genes, red scatter plots indicate 1262 significantly upregulated genes, and blue scatter plots indicate genes with no significant differences. p <0.05, log2FoldChange|>0. Figure 7 Bubble plot of GO functional enrichment of differentially upregulated genes in hUMSCs between the Control group and the 50 μAES group. These significantly upregulated genes are mainly related to mitochondrial biosynthesis and energy metabolism quality control, anti-inflammatory and antioxidant stress, cell adhesion / migration / secretion, angiogenesis and nerve growth. Figure 7 C: Clustering heatmap of the top 30 upregulated genes with differentially expressed genes in hUMSCs between the Control group and the 50 μAES group. Figure 7 From D to Figure 7 In step I: qRT-PCR was used to verify the expression of differentially expressed genes FGF7, FGFR2, ICAM5, TGFβ, laminin, and Integrinα1 in hUMSCs between the Control group and the 50 μAES group. Compared with the Control group, the 50 μAES group showed significantly upregulated expression, consistent with the transcriptome sequencing results.* p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, n =3.

[0060] like Figure 8 As shown, electrical stimulation can stimulate the differentiation potential of hUMSCs in the neural direction, and 1 mA of in vitro electrical stimulation can promote the differentiation of hUMSCs into neurons and oligodendrocytes. Figure 8 A: Fluorescent staining illustration shows the expression of TUJ1 and CC1 in hUMSCs without electrical stimulation (Control group), after electrical stimulation of hUMSCs at 50 μA, 1 mA, and 4 mA. TUJ1 (red), CC1 (green), and Hoe (blue) are shown. Scale bar = 100 μm. Figure 8B: Bar chart showing hUMSCs without electrical stimulation (Control group), hUMSCs after TUJ stimulation at 50μA, 1mA, and 4mA. + Cell proportion statistical analysis showed that, compared with the control group, the TUJ values ​​in the 50μA, 1mA, and 4mA groups were significantly higher. + The proportion of cells increased in all groups, with a significant upregulation in the 1 mA group.* p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, n =3. Figure 8 C: Bar chart showing the C1 levels of hUMSCs without electrical stimulation (Control group), hUMSCs after electrical stimulation with 50 μA, 1 mA, and 4 mA. + Cell proportion statistical analysis showed that, compared with the control group, the 50μA, 1mA, and 4mA groups had higher CC1 concentrations. + The proportion of cells increased in all groups, with a significant upregulation in the 1 mA group.* p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, n =3.

[0061] like Figures 9-11 As shown, EA can synergistically enhance the therapeutic effect of intrathecal infusion of hUMSCs. That is, intrathecal infusion of hUMSCs combined with EA treatment synergistically improves the microenvironment of the spinal cord injury area, promotes the survival of spinal cord neurons and their axonal growth, especially the regeneration of descending nerve fibers or the sprouting of collateral branches that cross the injury area to re-innervate the lumbar spinal cord motor neurons, thus promoting the recovery of motor function in the paralyzed hind limbs of beagle dogs.

[0062] Figure 9 The expression of NF and GFAP in the spinal cord of each group by immunofluorescence and Western blot is as follows: Figure 9 As shown. Figure 9 China A to Figure 9 D: Low-magnification images of the spinal cord injury area and surrounding NF and GFAP in the hUMSCs+EA group, hUMSCs group, EA group, and Control group. NF+ (red), GFAP+ (white), Hoe (blue). Scale bar = 1000 μm. a1-d5: High-magnification images of NF+ and GFAP+ in five regions of the spinal cord injury area in the hUMSCs+EA group, hUMSCs group, EA group, and Control group: cephalic end, cephalic-cephalic interface, injury area, caudal interface, and caudal end of the injury area. Scale bar = 20 μm. Figure 9E: Western blot results showed the expression of NF and GFAP proteins in the spinal cord injury area of ​​each group. Figure 9 In section e1: The bar charts show the gray-scale ratio of the NF protein bands in each group detected by Western blot and their statistical analysis results (mean ± SD). Figure 9 Table e2: Bar charts show the gray-scale ratios of GFAP protein bands in each group detected by Western blot and their statistical analysis results (mean ± SD). Compared with the hUMSCs+EA group, *p<0.05, **p<0.01, **p<0.01, n = 4; compared with the hUMSCs group, #p<0.05, n = 4; compared with the EA group, %p<0.05, n = 4.

[0063] Imaging assessments of beagle dogs after spinal cord surgery and treatment using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI), such as... Figure 10 As shown. Figure 10 Middle A1 to Figure 10 d1: Low and high magnification MRI images of beagle dogs 2 weeks after spinal cord surgery in each group. Figure 10 Middle A2 to Figure 10 D2: Low and high magnification MRI images of beagle dogs 3 months post-surgery for spinal cord injuries in each group; A3-D3: DTI images of beagle dogs 3 months post-treatment for spinal cord injuries in each group, with yellow and blue representing the cephalic and caudal spinal cord nerve fiber bundles in the injury area, respectively. Results are as follows: Figure 10 a1 to Figure 10 As shown in D1, the MRI imaging data clearly showed the condition of the postoperative spinal cord injury area, with high-density images and a cavity-like shape appearing in the T12 segment of the spinal cord. This result indicates the successful establishment of the beagle dog spinal cord constriction model. After treatment, MRI and diffusion tensor imaging were performed to assess the effect of human umbilical cord mesenchymal stem cell therapy combined with EA on promoting the structural recovery of the spinal cord injury. The results are as follows... Figure 10 a2 to Figure 10 As shown in Figure e2, the spinal cord injury areas in the hUMSCs+EA group, hUMSCs group, and EA group showed a reduction in high-density imaging area. In the untreated Control group, the high-density imaging area was slightly reduced, while cavitation-like structures were more pronounced. Figure 10 Middle A3 to Figure 10 The DTI results shown in D3 indicate that in the hUMSCs+EA group, the hUMSCs group, and the EA group, the nerve fibers regenerating at the head and tail ends of the spinal cord injury were closer together, and the interrupted area of ​​the injury zone was significantly shortened. In contrast, the distance between the head and tail ends of the injury was larger in the Control group. These results demonstrate that intrathecal infusion of hUMSCs combined with EA can promote structural recovery in the spinal cord injury area of ​​beagle dogs.

[0064] The recovery status of spinal motor function in each group of beagle dogs is as follows: Figure 11 As shown. Figure 11 A: Hind limb motor recovery status of Beagle dogs 3 months after spinal cord injury surgery in each group. Figure 11 Image B: Electrophysiological records of beagle dogs in each group 3 months after surgery. Figure 11 C to Figure 11 D: Statistical graph of the latency and peak of electrophysiology in each group of beagle dogs. Figure 11 E: Beagle behavioral Obly scores for each group. Compared with the hUMSCs+EA group, *p<0.05, **p<0.01, ***p<0.001, n=4; compared with the hUMSCs group, #p<0.05, #p<0.01, n=4; compared with the EA group, %p<0.05, n=4. Behavioral results ( Figure 11 China A and Figure 11 As shown in Figure E, before surgery, the Olby scores of all groups of beagles were 14, indicating no motor dysfunction. Three days post-surgery, the Olby scores of all groups were 0, indicating complete loss of function in both hind limbs. Behavioral assessments were conducted weekly thereafter, revealing significant recovery of voluntary motor function in both hind limbs starting one month post-surgery. The treated hUMSCs+EA group showed significant behavioral improvement compared to the untreated Control group. Three months post-surgery, the hUMSCs+EA, hUMSCs, and EA groups all demonstrated limb coordination, weight-bearing ability, and the ability to stand. However, compared to the hUMSCs and EA groups, the hUMSCs+EA group showed better coordination and stability in walking, while the untreated Control group only exhibited joint movement and was unable to support full weight. Figure 11 (A). Electrophysiological testing was performed to further understand the recovery of nerve conduction function. Results are as follows: Figure 11 China B to Figure 11 The results showed that 3 months post-surgery, the hUMSCs+EA group of beagle dogs exhibited shorter CMEP latency and larger peak-to-peak action potentials, while the control group showed longer CMEP latency, smaller peak-to-peak values, and difficulty in distinguishing the main wave morphology. These results indicate that human umbilical cord mesenchymal stem cells combined with EA treatment can promote the recovery of motor function in beagle dogs with spinal cord injury.

[0065] This application utilizes an experimental method involving in vitro electrical stimulation with different parameters to enhance the survival rate, secretory function, and neural differentiation potential of hUMSCs. This method is more conducive to identifying key therapeutic target genes for spinal cord injury repair using electroacupuncture combined with hUMSCs, thereby providing in vitro experimental evidence for the study of the mechanism of functional electrical stimulation of hUMSCs and the selection of electrical stimulation parameters for the treatment of spinal cord injury using electroacupuncture combined with hUMSCs.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells, characterized in that, Human umbilical cord mesenchymal stem cells are electrically stimulated, and the human umbilical cord mesenchymal stem cells and their secretory supernatant are collected for detection or in vitro experiments. The conditions for the electrical stimulation include: a current of 5 μA to 4 mA, an electric field strength of 0.625 mV / mm to 500 mV / mm, and a frequency of 20 Hz.

2. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 1, wherein the conditions for electrical stimulation include: The current ranges from 5 μA to 50 μA, the electric field strength ranges from 0.625 mV / mm to 6.25 mV / mm, and the frequency is 20 Hz.

3. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 1, wherein the conditions for electrical stimulation further include a duty cycle DR = 50%, a single electrical stimulation time of 30 min, and a continuous electrical stimulation time of 3 to 5 days.

4. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The electrical stimulation is performed using an external electrical stimulation device; the external electrical stimulation device and the waveform generator are directly connected. The external electrical stimulation device includes a conductive cell culture dish at the bottom and a culture dish lid with a conductive metal sheet at the top. The materials used to prepare the conductive cell culture dish at the bottom include ITO transparent conductive glass, quartz glass rings, and PDMS adhesive. The materials used to prepare the petri dish lid with a conductive metal sheet on top include acrylic, stainless steel, and ABS resin.

5. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 4, characterized in that, In the conductive cell culture dish at the bottom, a quartz glass ring is attached to the center of an ITO transparent conductive glass ring. The quartz glass ring and the ITO transparent conductive glass are bonded with PDMS adhesive. The ITO transparent conductive glass is connected to the positive / negative electrodes of the waveform generator. And / or, The petri dish lid with a conductive metal sheet on top is assembled from a 3D-printed acrylic transparent lid and a stainless steel electrode using ABS resin fixing posts.

6. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 4, characterized in that, In the culture dish lid with a conductive metal sheet on top, the cell culture medium contact surface of the stainless steel electrode is gold-plated using a sputtering gold process, and the other side is welded with a stainless steel cylinder for connecting the negative / positive terminals of the waveform generator.

7. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The cell density of the human umbilical cord mesenchymal stem cells is 70%~80%.

8. The method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The number of human umbilical cord mesenchymal stem cells is approximately 1~5×10⁻⁶. 5 indivual.

9. The application of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in any one of claims 1 to 8 in improving the survival, secretory function and neural differentiation potential of human umbilical cord mesenchymal stem cells.

10. The application of the method for in vitro electrical stimulation of human umbilical cord mesenchymal stem cells as described in any one of claims 1 to 8 in the treatment of spinal cord injury.

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