Umbilical cord mesenchymal stem cells screened based on igf1r expression and application thereof in treatment of cerebral ischemic injury and acute myocardial infarction

Abstract

This invention discloses an umbilical cord mesenchymal stem cell selection method based on IGF1R expression and its application in the treatment of ischemic brain injury (stroke) and acute myocardial infarction (AMI), belonging to the field of biomedical technology. This invention screens and validates IGF1R... + Mesenchymal stem cells, as a highly effective therapeutic subset, significantly improved neurological function after cerebral ischemia-reperfusion injury and cardiac function recovery in acute myocardial infarction. IGF1R showed a positive expression rate of 85%. + Mesenchymal stem cells, compared with cells with 30% and 60% positive expression rates and the control group, significantly improved the neurological deficit symptoms in rats with cerebral ischemia-reperfusion injury and the recovery of cardiac function in rats with acute myocardial infarction. They also increased the number of vertical movements and the duration of vertical movement in rats, demonstrating excellent neuroregenerative potential and significant myocardial repair and regeneration capabilities.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an umbilical cord mesenchymal stem cell screened based on IGF1R expression and its application in the treatment of ischemic brain injury and acute myocardial infarction (AMI). Background Technology

[0002] Stroke is one of the leading causes of death and disability worldwide, and cerebral ischemia-reperfusion injury is a key pathological step in the development of stroke. When the blood supply to the brain is interrupted, causing ischemic injury, the process of restoring blood flow further exacerbates oxidative stress, inflammatory response, and neuronal apoptosis in brain tissue. Ultimately, this leads to severe neurological deficits such as limb motor dysfunction and cognitive decline, placing a heavy medical burden and economic pressure on patients' families and society.

[0003] The main cause of acute myocardial infarction is the sudden interruption of coronary blood flow, leading to irreversible necrosis of myocardial cells due to prolonged hypoxia. Common predisposing factors include atherosclerosis, which is the long-term accumulation of fatty and cholesterol plaques on the blood vessel walls, narrowing the lumen of the blood vessels and causing thrombus formation. When the plaque suddenly ruptures, it triggers platelet aggregation to form a thrombus, completely blocking the blood vessel. Risk factors include hypertension, hyperglycemia, hyperlipidemia (the "three highs"), smoking, obesity, and family history.

[0004] Currently, clinical treatment options for cerebral ischemia-reperfusion injury are limited. Thrombolytic therapy and endovascular interventional therapy have strict time windows, only applicable within hours of onset, meaning most patients miss the optimal treatment window and thus do not benefit. Furthermore, traditional drug therapies, such as neuroprotective agents, are not ideal in improving long-term neurological outcomes and struggle to fundamentally repair damaged brain tissue and neural circuits. In acute myocardial infarction, current treatments (such as cardiac catheterization and thrombolytic agents) are essentially like "opening a pipe," focusing on restoring blood flow, but often ineffective against already necrotic myocardial cells. Treatment currently faces a formidable challenge.

[0005] In recent years, mesenchymal stem cells (MSCs) have shown potential therapeutic value in the field of neural regeneration and repair due to their self-renewal, multi-lineage differentiation, and immunomodulatory properties. However, MSCs are diverse in origin and highly heterogeneous, with significant differences in therapeutic efficacy among different subpopulations, leading to unstable clinical translational results and poor reproducibility. Current technologies have not yet clearly identified key surface biomarkers for effectively screening highly effective therapeutic subpopulations, nor are there systematic reviews addressing functional differences among subpopulations of MSCs from different sources. This limits the precise application and efficacy optimization of stem cell therapy in cerebral ischemia-reperfusion injury and acute myocardial infarction. Summary of the Invention

[0006] The purpose of this invention is to provide umbilical cord mesenchymal stem cells screened based on IGF1R expression and their application in the treatment of ischemic brain injury and acute myocardial infarction (AMI), so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is an IGF1R + The application of mesenchymal stem cells in the preparation of drugs for treating cerebral ischemia-reperfusion injury and acute myocardial infarction (AMI), wherein the IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 30% to 85%.

[0008] The second technical solution of this invention is a drug for treating cerebral ischemia-reperfusion injury (Stroke) and acute myocardial infarction (AMI), comprising IGF1R. + Mesenchymal stem cells, The IGF1R + The method for preparing mesenchymal stem cells includes the following steps: (1) Human mesenchymal stem cells were cultured for 4-8 generations and immunophenotypic identification was performed by flow cytometry. The identification indicators included CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR; (2) The cells identified in step (1) were sorted by flow cytometry to obtain the IGF1R. + Mesenchymal stem cells.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention screens and verifies IGF1R + Mesenchymal stem cells, as a highly effective therapeutic subgroup, significantly improved the recovery of neurological and cardiac function after cerebral ischemia-reperfusion injury and acute myocardial infarction (AMI). IGF1R showed a positive expression rate of 85%. + Mesenchymal stem cells, compared to cells with 30% and 60% positive expression rates and the control group, significantly improved neurological deficits in rats with cerebral ischemia-reperfusion injury and cardiac function recovery in rats with acute myocardial infarction. They also increased the number and duration of vertical movement and the area of ​​myocardial infarction repair, demonstrating excellent neurological and cardiac regenerative potential. This invention establishes a standardized IGF1R... +This invention relates to a process for culturing, identifying, and sorting mesenchymal stem cells, which solves the problems of uneven activity and unstable therapeutic effects associated with traditional unsorted mesenchymal stem cells. The invention also relates to IGF1R derived from umbilical cord, adipose tissue, and bone marrow. + Mesenchymal stem cells are readily available and can be administered via intravenous injection, making the procedure simple and minimally invasive. This provides a new and effective treatment option for stroke and acute myocardial infarction, and has high clinical translational value. Attached Figure Description

[0010] Figure 1 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical movement frequency after umbilical cord mesenchymal stem cell therapy.

[0011] Figure 2 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical mobility after treatment with adipose-derived mesenchymal stem cells.

[0012] Figure 3 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical mobility after treatment with bone marrow-derived mesenchymal stem cells.

[0013] Figure 4 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical movement time after umbilical cord mesenchymal stem cell therapy.

[0014] Figure 5 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical movement time after treatment with adipose-derived mesenchymal stem cells.

[0015] Figure 6 Following cerebral ischemia-reperfusion injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of improved vertical movement time after treatment with bone marrow-derived mesenchymal stem cells.

[0016] Figure 7 Following acute myocardial infarction (AMI) injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of myocardial infarction area repair after umbilical cord mesenchymal stem cell therapy.

[0017] Figure 8Following acute myocardial infarction (AMI) injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of myocardial infarction area repair after treatment with adipose-derived mesenchymal stem cells.

[0018] Figure 9 Following acute myocardial infarction (AMI) injury in rats, treatment with 30%, 60%, and 85% IGF1R was initiated. + Results of myocardial infarction area repair after bone marrow-derived mesenchymal stem cell therapy. Detailed Implementation

[0019] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0020] This invention provides an IGF1R + The application of mesenchymal stem cells in the preparation of drugs for treating cerebral ischemia-reperfusion injury and acute myocardial infarction (AMI), wherein the IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 30% to 85%.

[0021] In some specific implementations, the human mesenchymal stem cells are selected from at least one of umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

[0022] In some specific implementations, the IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 60% to 85%.

[0023] In some specific implementations, the IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 85%.

[0024] In some specific implementations, the IGF1R + The method for preparing mesenchymal stem cells includes the following steps: (1) Human mesenchymal stem cells were cultured for 4-8 generations and immunophenotypic identification was performed by flow cytometry. The identification indicators included CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR; (2) The cells identified in step (1) were sorted by flow cytometry to obtain the IGF1R.+ Mesenchymal stem cells.

[0025] This invention also provides a drug for treating cerebral ischemia-reperfusion injury, including IGF1R. + Mesenchymal stem cells, The IGF1R + The method for preparing mesenchymal stem cells includes the following steps: (1) Human mesenchymal stem cells were cultured for 4-8 generations and immunophenotypic identification was performed by flow cytometry. The identification indicators included CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR; (2) The cells identified in step (1) were sorted by flow cytometry to obtain the IGF1R. + Mesenchymal stem cells.

[0026] Example 1 1. Preparation, immunophenotyping, and cell sorting of human mesenchymal stem cells (MSCs) Human mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) and included umbilical cord mesenchymal stem cells (UMSCs, ATCC PCS 500-010), adipose-derived mesenchymal stem cells (ADSCs, ATCC PCS 500-011), and bone marrow-derived mesenchymal stem cells (BMSCs, ATCC PCS 500-012).

[0027] The mesenchymal stem cells were cultured in NutriStem® MSC XF medium supplemented with NutriStem® XF supplement mixture (Sartorius, Germany), 5% UltraGRO™-Advanced-PURE cell culture supplement (AventaCell Biomedical, USA) and antibiotics at 37°C and 5% carbon dioxide.

[0028] After seeding, cells were left to stand for 5-7 days to allow them to migrate out of the tissue block (refer to Scientific Report 2016;6:32595). After 4-8 passages of culture, the morphology of umbilical cord mesenchymal stem cells became homogeneous and spindle-shaped (refer to Scientific Report 2016;6:32595).

[0029] Flow cytometry was used to identify specific surface molecules of human mesenchymal stem cells: Cells were digested with TrypLE™ Select enzyme (Gibco, USA), washed with PBS, and then fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled antibodies (BD Biosciences, USA) were added, including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR; data were then analyzed using a Beckman Coulter flow cytometer and FlowJo v.7.6 software.

[0030] During cell sorting, the method was followed as previously published (Journal of Neuroscience, 2003; Vol. 23: 240-251) (this article could not be found), using FACSTAR. + Flow cytometry (Beckman Coulter) for the purification of IGF1R + Mesenchymal stem cells (UMSCs, ADSCs, and BMSCs) showed positive expression rates of insulin-like growth factor 1 receptor (IGF1R) of 30%, 50%, and 80%, respectively. Trypan blue rejection assays confirmed that the cell viability after sorting was approximately 96%.

[0031] 2. Animal models of cerebral ischemia-reperfusion and acute myocardial infarction (AMI) in rats Adult male Sprague-Dawley rats (weighing 250-300g) were selected, and a three-vessel ligation method was used to establish the model. All surgical procedures, animal experimental protocols, and methods followed institutional guidelines and were approved by the Animal and Clinical Research Ethics Committee of China Medical University (Taichung, Taiwan).

[0032] Rats were anesthetized by intraperitoneal injection of chloral hydrate (0.4 g / kg). Following a slightly modified method described previously, the right middle cerebral artery (MCA) and both common carotid arteries (CCAs) were ligated. The bilateral common carotid arteries were clamped using non-invasive arterial clips. Under a surgical microscope, a 2×2 mm cranial window was created at the junction of the zygomatic arch and squamous bone. The right middle cerebral artery was ligated using 10-0 nylon sutures. Cortical blood flow in the anesthetized rats was continuously monitored using a laser Doppler flowmeter (Periflux system, PF-5010, Sweden): a 1 mm diameter hole was drilled in the right frontoparietal lobe region, and a detector was implanted; a 0.45 mm diameter probe was stereotactically positioned in the cortex (1.3 mm posterior to the anterior fontanelle, 2.8 mm lateral to it, and 1.0 mm subdurally). After 90 minutes of ischemia, the middle cerebral artery sutures and the common carotid artery clips were removed, and blood perfusion was restored. During anesthesia, core body temperature was monitored using a thermistor probe and maintained at 37°C using a heating pad; after anesthesia recovery, body temperature was maintained at 37°C using a heating lamp.

[0033] Adult male Sprague-Dawley (SD) rats (weighing 200-250 g) were used to establish an AMI model by ligating the left anterior descending coronary artery (LAD) according to the method described in previous literature (Nature Protocols, 2006; 1: 1596-1609).

[0034] The specific procedures were as follows: Rats were anesthetized with a mixture of 2% isoflurane and 100% oxygen. Artificial ventilation was performed using a small animal ventilator (SN-480-7, Japan), with a tidal volume of 1 mL / 100 g and a respiratory rate of 80 breaths / minute. A thoracotomy was performed on the left side at the 4th-5th intercostal space, exposing the surgical field using a rib retractor (MY-9454S, Japan). The left lung was gently compressed with a small gauze pad soaked in physiological saline to cause collapse. After opening the pericardium, the myocardium was ligated 1-2 mm below the atrioventricular groove using 6-0 polypropylene sutures (Ethicon, UK). After ligation, the lung was re-expanded, and the chest was closed layer by layer. After myocardial ligation, echocardiography confirmed the success of the surgery, and stem cell therapy was administered within 30 minutes. In the sham surgery group, all surgical procedures were the same as in the model group, except that the coronary arteries were not ligated.

[0035] This research protocol was approved by the Animal Experiment Ethics Committee of China Medical University, Taiwan.

[0036] 3. IGF1R + Intravenous injection of mesenchymal stem cells After cell sorting, the cells will be processed by FACSTAR. + IGF1R obtained by flow cytometry immunosorting +Cells (IGF1R positive expression rates of 30%, 50%, and 80%) were cultured in a humid environment at 37°C with 5% carbon dioxide and 95% air. 1×10⁻⁶ cells were added. 6 IGF1R + UMSCs, ADSCs, or BMSCs were suspended in 100 μL of PBS and injected at a dose of 1 × 10⁻⁶ using a 26-gauge syringe. 6 One stem cell injection.

[0037] The experiment was divided into four groups: IGF1R + (30%-UMSCs, ADSCs or BMSCs) group, IGF1R + (60%-UMSCs, ADSCs, or BMSCs) group, IGF1R + (85%-UMSCs, ADSCs or BMSCs) group and control group (only injected with solvent).

[0038] 4. Neurobehavioral assessment, myocardial histology examination, and infarct area measurement Behavioral assessments were conducted 5 days before cerebral ischemia and 1, 7, 14, and 28 days after cell transplantation to assess the spontaneous activity of each rat. Baseline test scores were recorded to calibrate the results after cerebral ischemia. Behavioral recordings were performed for approximately 2 hours using a VersaMax animal activity monitoring system (Accuscan Instruments, Columbus, Ohio, USA). This system contains 16 horizontal infrared sensors and 8 vertical infrared sensors, with the vertical sensors positioned 10 cm from the bottom of the testing chamber. The number of times the rat blocked the light beam while moving within the chamber was counted as the amount of movement activity. Two vertical indicators were calculated over the 2-hour period: (i) the number of vertical movements; and (ii) the duration of vertical movement.

[0039] Experimental rats were sacrificed on days 3 and 28 after AMI surgery, under heavy anesthesia with chloral hydrate (0.4 g / kg, intraperitoneal injection). Three rats without LAD ligation were selected as the normal control group. Following the method in previous literature (Circulation, 2012; 126: S46-53), rat hearts were fixed in 4% paraformaldehyde and then immersed in 30% sucrose solution for 3 days. The tissue blocks were serially sectioned along the coronal plane using a cryostat, with a section thickness of 6 μm, stained with hematoxylin and eosin (H&E), and observed and analyzed under an optical microscope (Nikon, E600).

[0040] Following the method described in the literature (Am J Physiol Heart Circ Physiol, 2006; 291: H1972-1977), sections at different levels (apex, mid-left ventricular segment, and base) along the long axis of the heart were stained with Masson's trichrome (Sigma). The infarct area was calculated using ImageJ image analysis software (National Institutes of Health, NIH). Twenty-eight days after AMI, the infarct area was expressed as a percentage of the left ventricular circumference based on the Masson's trichrome staining results. Objective: To validate IGF1R. + The effect of mesenchymal stem cell (MSC) transplantation on the amelioration of ischemic myocardial infarction area in rats with acute myocardial infarction (AMI), and a semi-quantitative analysis of the infarct area after surgery.

[0041] 5. Experimental Results: Includes cerebral ischemia-reperfusion injury (Stroke) and acute myocardial infarction (AMI). To verify IGF1R + To investigate whether mesenchymal stem cell transplantation can improve neurobehavioral function in stroke-affected rats, the degree of neurological deficit (spontaneous movement ability: vertical movement frequency and duration) before and after stroke was assessed. Rats were divided into four groups for treatment intervention, including IGF1R... + (30%-UMSCs, ADSCs or BMSCs) group, IGF1R + (60%-UMSCs, ADSCs, or BMSCs) group, IGF1R + The study included groups of rats with 85%-UMSCs, ADSCs, or BMSCs and a control group to assess behavioral recovery in stroke-affected rats.

[0042] The results showed that IGF1R + The recovery of rats treated with (85%-UMSCs, ADSCs, or BMSCs) was superior to that of rats treated with IGF1R. + (60%-UMSCs, ADSCs or BMSCs) treatment group ( Figure 1-6 (n=6, *P<0.01, P<0.05); and IGF1R + Compared with the (30%-UMSCs, ADSCs, or BMSCs) group and the control group, IGF1R + (85%) group and IGF1R + (60%) of the groups showed significant improvement in neurological deficits. Figure 1-6 (n=6, *P<0.01, P<0.05). The above results indicate that IGF1R + (85% - UMSCs, ADSCs or BMSCs) have superior neuroregenerative potential.

[0043] The rats were randomly divided into four groups: IGF1R + (30%) - MSCs group (human umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow mesenchymal stem cells), IGF1R + (60%) - MSCs group, IGF1R + (85%)-MSCs group and solvent control group. Results showed that IGF1R + The myocardial infarction area in the (85%)-MSCs group was significantly smaller than that in the IGF1R group. + (60%) - MSCs group ( Figure 7-9 (n=6, *P<0.01, P<0.05). Simultaneously, with IGF1R... + Compared with the (30%)-MSCs group and the control group, IGF1R + (85%) - MSCs group and IGF1R + (60%) - The infarct area in the MSCs group was significantly reduced. Figure 7-9 (n=6, *P<0.01, P<0.05). The above results indicate that IGF1R + (85%)-MSCs have a significant ability to repair and regenerate ischemic myocardium.

[0044] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An IGF1R + The application of mesenchymal stem cells in the preparation of drugs for treating cerebral ischemia-reperfusion injury and acute myocardial infarction is characterized by, The IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 30% to 85%.

2. The application according to claim 1, characterized in that, The human mesenchymal stem cells are selected from at least one of umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

3. The application according to claim 1, characterized in that, The IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 60% to 85%.

4. The application according to claim 1, characterized in that, The IGF1R + Mesenchymal stem cells are human mesenchymal stem cells with an IGF1R positive expression rate of 85%.

5. The application according to claim 1, characterized in that, The IGF1R + The method for preparing mesenchymal stem cells includes the following steps: (1) Human mesenchymal stem cells were cultured for 4-8 generations and immunophenotypic identification was performed by flow cytometry. The identification indicators included CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR; (2) The cells identified in step (1) were sorted by flow cytometry to obtain the IGF1R. + Mesenchymal stem cells.

6. A drug for treating cerebral ischemia-reperfusion injury and acute myocardial infarction, characterized in that, Including IGF1R + Mesenchymal stem cells, The IGF1R + The method for preparing mesenchymal stem cells includes the following steps: (1) Human mesenchymal stem cells were cultured for 4-8 generations and immunophenotypic identification was performed by flow cytometry. The identification indicators included CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR; (2) The cells identified in step (1) were sorted by flow cytometry to obtain the IGF1R. + Mesenchymal stem cells.

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