Use of umbilical artery paracrine stem cell spheroids in the preparation of a medicament for treating ischemic cardiomyopathy

Abstract

The application discloses application of umbilical cord arterial paramagnetic stem cell spheroids in preparation of a medicine for treating ischemic cardiomyopathy, and the umbilical cord arterial paramagnetic stem cell spheroids can significantly improve heart function, reduce myocardial infarction area, reduce myocardial fibrosis and increase microvessel density when being administered at 3 days after myocardial infarction, and the effect is long-lasting, thereby providing an effective treatment option for patients who miss the reperfusion treatment time window. The preparation method is simple and controllable, the cell spheroids are uniform in size and high in survival rate, the cell spheroids are suitable for standardized production and clinical transformation, and the application has a good treatment product development prospect.

Description

Technical Field

[0001] This invention relates to biomedical technology, specifically to the application of a para-umbilical artery stem cell sphere in the preparation of a drug for treating ischemic cardiomyopathy, as well as a method for preparing the para-umbilical artery stem cell sphere and a therapeutic drug containing the cell sphere. Background Technology

[0002] Acute myocardial infarction (AMI) is a cardiovascular emergency caused by acute occlusion of the coronary arteries, leading to myocardial ischemia and necrosis. Although thrombolytic therapy and percutaneous coronary intervention (PCI) significantly reduce acute-phase mortality, a considerable proportion of patients cannot receive timely reperfusion therapy due to delayed medical attention, limited medical resources, or socioeconomic factors. Delayed reperfusion not only offers limited benefits but may also exacerbate adverse left ventricular remodeling, ultimately leading to ischemic cardiomyopathy and heart failure. Therefore, developing effective regenerative therapy strategies is of significant clinical importance for patients with acute myocardial infarction who do not receive timely reperfusion therapy. Angiogenesis is a core component of myocardial repair after myocardial infarction. Adequate angiogenesis improves oxygen and nutrient supply, limits infarct expansion, reduces adverse remodeling, and protects cardiac function. Conversely, insufficient or dysregulated angiogenesis leads to scar expansion and progressive cardiac dysfunction.

[0003] The umbilical cord consists of one umbilical vein and two umbilical arteries, embedded in Wharton's jelly, from which different mesenchymal and pericyte populations can be isolated. Besides Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), para-umbilical vein stem cells (UCV-PSCs) and para-umbilical artery stem cells (UCA-PSCs) can also be isolated from the umbilical arteries. UCA-PSCs express classic mesenchymal stem cell markers (CD13, CD29, CD44, CD90, CD105) and pericyte-related marker CD146, exhibiting multi-lineage differentiation potential. However, stem cell therapy for myocardial infarction faces limitations such as low post-transplant cell survival rate, short retention time, and insufficient paracrine activity. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an application of para-umbilical artery stem cell spheres in the preparation of drugs for treating ischemic cardiomyopathy, in order to solve the technical problems of low cell survival rate, highly invasive administration methods, and short-lasting therapeutic effects in existing stem cell therapy for myocardial infarction.

[0005] Technical solution: A para-umbilical artery stem cell sphere, wherein the para-umbilical artery stem cell sphere is formed by three-dimensional culture of para-umbilical artery stem cells (UCA-PSCs) and has a diameter of 100-200μm.

[0006] A method for preparing para-umbilical artery stem cell spheres includes the following steps: Para-umbilical artery stem cell culture: Para-umbilical artery stem cells were obtained by culturing them in complete culture medium at 30-40°C and 1-10% CO2. Preparation of para-arterial stem cell spheres: Adjust the concentration of the single-cell suspension to 1×10⁻⁶. 5 ~10×10 5 Cells were seeded at a density of 100-200 μm / mL in ultra-low adsorption culture plates and cultured until they spontaneously aggregated to form cell spheroids with a diameter of 100-200 μm. Preferably, the complete culture medium was low-glucose DMEM medium, 10-15% fetal bovine serum, and 0.5-1.5% penicillin-streptomycin antibiotics.

[0007] An injection solution containing para-umbilical artery stem cell spheres.

[0008] A method for preparing a peri-umbilical artery stem cell sphere injection solution includes the following steps: Para-umbilical artery stem cell culture: Para-umbilical artery stem cells were obtained by culturing them in complete culture medium at 30-40°C and 1-10% CO2. Preparation of para-arterial stem cell spheres: Adjust the concentration of the single-cell suspension to 1×10⁻⁶. 5 ~10×10 5 Cells / mL were seeded into ultra-low adsorption culture plates and cultured until the cells spontaneously aggregated to form cell spheres with a diameter of 100-200 μm. Preparation of para-umbilical artery stem cell sphere injection solution: Collect cell spheres, wash with physiological saline, and finally prepare the para-umbilical artery stem cell sphere injection solution using 1-10% human serum albumin injection solution. Preferably, the complete culture medium is low-glucose DMEM medium, 10-15% fetal bovine serum, and 0.5-1.5% penicillin-streptomycin antibiotics.

[0009] A method for detecting para-umbilical artery stem cell sphere injection, including cell viability and CD146 expression level.

[0010] The detection method for the para-invasive umbilical artery stem cell sphere injection solution is as follows: the cell viability is not less than 90% and the CD146 expression level is significantly higher than that of two-dimensional cultured cells to be considered qualified; otherwise, it is unqualified. The significant difference in CD146 expression level compared to two-dimensional cultured cells is determined by Western blot or qPCR detection with statistical significance (P ≤ 0.05).

[0011] The use of the para-umbilical artery stem cell sphere or the injection solution of the para-umbilical artery stem cell sphere in the preparation of drugs for treating ischemic cardiomyopathy.

[0012] The aforementioned application, wherein the ischemic cardiomyopathy includes acute myocardial infarction, post-myocardial infarction heart failure, or chronic ischemic cardiomyopathy.

[0013] The application described herein includes the use of an injectable form of the drug.

[0014] A pharmaceutical composition comprising para-umbilical artery stem cell spheres as described above and a pharmaceutically acceptable carrier.

[0015] The application of a para-umbilical artery stem cell sphere in the preparation of a drug for treating ischemic cardiomyopathy, wherein the para-umbilical artery stem cell sphere is formed by three-dimensional culture of para-umbilical artery stem cells (UCA-PSCs) and has a diameter of 100-200 μm. Preferably, the para-umbilical artery stem cell sphere highly expresses CD146 and stromal cell-derived factor 4 (SDF4).

[0016] The drug is administered via subcutaneous injection.

[0017] Preferably, the para-intra-arterial stem cell sphere exerts its therapeutic effect through the following dual paracrine mechanisms: (1) secreting SDF4 to activate the CXCR4 / AKT / ERK signaling pathway, directly promoting the proliferation, migration and lumen formation of vascular endothelial cells; (2) releasing soluble CD146 (sCD146) to inhibit the expression of neutrophil-derived S100A8 and S100A9, optimizing the pro-angiogenic microenvironment and reducing the inhibitory effect of inflammation on angiogenesis.

[0018] Preferably, the ischemic cardiomyopathy includes acute myocardial infarction, post-myocardial infarction heart failure, or chronic ischemic cardiomyopathy. Preferably, the drug prepared from the para-umbilical artery stem cell spheres is a liquid injectable preparation with a cell viability of not less than 90%.

[0019] Preferably, the drug is administered subcutaneously.

[0020] Preferably, the drug is administered 3 days after myocardial infarction, and the injection dose is at least 1 × 10⁻⁶ cells. 6 indivual.

[0021] Preferably, the evaluation method for the drug prepared from the para-umbilical artery stem cell spheres after treatment of ischemic cardiomyopathy includes one or more of the following: A. Monitoring changes in recipient cardiac function indicators, including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS), left ventricular end-systolic diameter (LVIDs), and left ventricular anterior wall systolic thickness (LVAWs); B. Observing changes in the recipient's myocardial infarction area; C. Observing changes in the recipient's myocardial fibrosis degree; D. Observing changes in the recipient's myocardial microvessel density; E. Monitoring changes in the levels of SDF4, sCD146, S100A8, and S100A9 in the recipient's serum; F. Monitoring changes in the phosphorylation levels of AKT and ERK1 / 2 in the recipient's myocardial tissue.

[0022] A method for preparing para-umbilical artery stem cell spheres includes the following steps: Step 1: Para-umbilical artery stem cell culture: Using low-glucose DMEM medium, 12% fetal bovine serum, and 1% penicillin-streptomycin as the complete culture medium, para-umbilical artery stem cells are obtained by culturing at 37°C and 5% CO2; Step 2: Preparation of para-umbilical artery stem cell spheres: The concentration of the single-cell suspension is adjusted to 4×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 500 μL / mL in 24-well ultra-low adsorption plates and cultured for 3 days to spontaneously aggregate into cell spheroids with a diameter of 100-200 μm. Step 3: Preparation of para-umbilical artery stem cell spheroid injection solution: Cell spheroids were collected, washed at least three times with physiological saline, and finally prepared using 5% human serum albumin injection solution to form the para-umbilical artery stem cell spheroid injection solution. Step 4: Cell viability and CD146 expression levels were detected. Cell viability was detected using AO / PI staining, and CD146 protein expression was detected using immunofluorescence staining, Western blot, or flow cytometry to ensure that cell viability was not less than 90% and that CD146 expression level was significantly higher than that of two-dimensional cultured cells. The significantly higher CD146 expression level than two-dimensional cultured cells was statistically significant (P ≤ 0.05) as determined by Western blot or qPCR.

[0023] Preferably, the ultra-low adsorption culture plate in step two is an ultra-low adsorption 24-well culture plate, the culture time is 3 days, and the average diameter of the formed cell spheres is 100-200 μm.

[0024] Preferably, the cell concentration in the para-umbilical artery stem cell sphere injection solution in step three is 5 × 10⁻⁶ cells / mL. 6 The injection volume is 200 μL, with the number of cells / mL being 1.

[0025] A drug for treating ischemic cardiomyopathy contains para-umbilical artery stem cell spheres as described above.

[0026] Preferably, the drug further comprises a pharmaceutically acceptable carrier, wherein the carrier is a 5% human serum albumin injection.

[0027] Preferably, the drug is a ready-to-use injectable formulation, in which cell spheres are uniformly dispersed in a liquid carrier.

[0028] In this invention, we fabricated para-umbilical artery stem cells into stem cell spheres and found that subcutaneous transplantation of these spheres was more effective in treating ischemic cardiomyopathy. When comparing the total number of cells used in treatment (two-dimensional and three-dimensional), the total number of cells (2D and 3D) was the same; however, because dozens or hundreds of cells were aggregated together, the individual cell spheres appeared larger. Experiments also demonstrated that, when using the same number of cells, subcutaneous injection of para-umbilical artery stem cells versus para-umbilical artery stem cell spheres resulted in better therapeutic effects and less trauma.

[0029] The treatment goals for ischemic cardiomyopathy are myocardial function and prognosis (LVEF, heart failure readmission, death), while the core goals for lower limb ischemia are walking ability and limb events. Therefore, although both are ischemic diseases, their mechanisms and endpoints are completely different, and treatment plans or indications cannot be simply extrapolated. For example, cilostazol can improve intermittent lower limb claudication and increase walking distance, but its instructions clearly state that it is contraindicated in heart failure. Dipyridamole can be used for symptomatic treatment of intermittent lower limb claudication, but ischemic cardiomyopathy is not an indication.

[0030] Beneficial effects: Compared with the prior art, the advantages of this invention are: (1) This invention is the first to apply para-umbilical artery stem cell spheres to the treatment of ischemic cardiomyopathy. Through three-dimensional spherical culture, the angiogenesis-promoting ability and paracrine activity of the cells are significantly enhanced. After subcutaneous implantation, a long-term and continuous cardioprotective effect can be achieved. (2) This invention uses subcutaneous injection to avoid the invasiveness and arrhythmia risk of intramyocardial injection, as well as the problems of pulmonary first-pass effect and low cell retention rate of intravenous injection. It has the advantages of being minimally invasive, safe, and easy to operate, and patients have high compliance. (3) This invention reveals the dual paracrine mechanism of para-umbilical artery stem cell spheres in the treatment of ischemic cardiomyopathy: on the one hand, it directly promotes angiogenesis through the SDF4 / CXCR4 / AKT / ERK signal axis, and on the other hand, it optimizes the microenvironment by inhibiting the S100A8 / A9 inflammatory axis through sCD146. The mechanism is clear and provides a theoretical basis for precision treatment. (4) The umbilical artery para-stem cell spheres of the present invention can significantly improve cardiac function, reduce the infarct area, alleviate myocardial fibrosis, and increase microvascular density when administered 3 days after myocardial infarction. The effect is long-lasting (lasting for at least 10 weeks), providing an effective treatment option for patients who have missed the reperfusion therapy window. (5) The preparation method of the present invention is simple and controllable, the cell spheres are uniform in size and have high viability, and are suitable for standardized production and clinical translation, with good prospects for the development of "off-the-shelf" therapeutic products. Attached Figure Description

[0031] Figure 1The images show the preparation and characterization of para-umbilical artery stem cell spheres; (A) Morphology of two-dimensional cultured UCA-PSCs; morphology of three-dimensional cultured UCA-PSC spheres; statistical distribution of UCA-PSC sphere diameter; (B) Western blot detection of CD146 protein expression; (C) Quantitative analysis of CD146 protein expression; (D) Quantitative analysis of CD146 mRNA expression.

[0032] Figure 2 Image showing the expression of pro-angiogenic factors in para-umbilical artery stem cell spheres; (AE) mRNA expression levels of angiogenesis-related genes (ANG, EGF, IGF, IL6, PLGF);

[0033] Figure 3 Analysis of cardiac function improvement in patients with myocardial infarction treated with para-umbilical artery stem cell spheres; (A) Quantitative analysis of cardiac function parameters, including left ventricular ejection fraction (EF%), left ventricular fractional shortening (FS%), left ventricular end-systolic diameter (LVIDs), and left ventricular anterior wall systolic thickness (LVAWs).

[0034] Figure 4 Analysis of angiogenesis promoted by para-umbilical artery stem cell spheres after myocardial infarction; (A) CD31 immunofluorescence staining of myocardial tissue showing microvessel density; (B) Quantitative analysis of CD31 fluorescence intensity in the infarcted area; (C) Quantitative analysis of CD31 fluorescence intensity in the marginal area;

[0035] Figure 5 Mechanism analysis of para-umbilical artery stem cell spheres for myocardial infarction; (A) Transcriptomics GO enrichment analysis (angiogenesis-related biological processes); (B) KEGG pathway enrichment analysis; (C) Serum proteomics volcano plot showing differentially expressed proteins; (DE) Correlation analysis of serum sCD146 with S100A8 and S100A9 levels. Detailed Implementation

[0036] Example 1 Step 1: Para-arterial umbilical cord stem cell culture: After thawing frozen para-arterial umbilical cord stem cells, they were seeded into T75 culture flasks. The culture medium was low-glucose DMEM + 12% fetal bovine serum + 1% penicillin-streptomycin antibiotics, and the flasks were incubated at 37°C with 5% CO2. 2 Cultured in a saturated humidity incubator. Observe cell adhesion 24 hours after inoculation, remove suspended cells and replace with fresh complete culture medium, then change the culture medium every 2 days thereafter; when the cell confluence reaches about 80-90%, digest with Tryple for 2 minutes, gently tap the flask wall to detach the cells and add complete culture medium to stop digestion, passage at a 1:3 ratio, and continue culturing to the logarithmic growth phase for cell spheroid preparation.

[0037] Step 2: Preparation of para-umbilical artery stem cell spheres: After digestion, logarithmically growing cells were resuspended in complete culture medium. The cells were counted using an automated cell counter, and the concentration of the single-cell suspension was adjusted to 4 × 10^5 cells / mL. The cell suspension was seeded into 24-well ultra-low adsorption plates at 500 μL per well. The plates were gently shaken to distribute the cells evenly, avoiding the formation of air bubbles. The plates were then incubated statically at 37°C and 5% CO2 for 3 days without changing the culture medium. The spontaneous aggregation of cells was observed to form cell spheres with a diameter of approximately 100–200 μm, clear boundaries, and regular morphology.

[0038] Step 3: Preparation of para-umbilical artery stem cell sphere injection solution: Gently collect the cell sphere suspension with a pipette, centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Resuspend the spheres in sterile physiological saline and wash, repeating at least three times to remove residual culture medium components. Finally, resuspend the cell spheres in 5% human serum albumin injection solution to the required concentration to prepare the para-umbilical artery stem cell sphere injection solution for later use.

[0039] Step 4: Take a portion of the cell spheroid sample, digest it into single cells, and use the trypan blue exclusion method to detect cell viability, requiring a viability of not less than 90%. At the same time, use Western blotting and qPCR to detect CD146 expression level. With two-dimensional cultured cells as a control, the expression of CD146 in cells derived from cell spheroids was significantly increased (statistically significant, P ≤ 0.05), indicating three-dimensional spheroid formation.

[0040] Characterization diagram of the prepared para-umbilical artery stem cell spheres is shown below. Figure 1 As shown, the cell spheroids had a regular shape and concentrated diameter distribution, and CD146 expression was significantly higher than that of two-dimensional cultured cells. The pro-angiogenic factors were also detected, and the results are as follows: Figure 2 Most pro-angiogenic factors (such as Ang, EGF, IL-6, PLGF, etc.) are upregulated in the cell spheres, suggesting that the three-dimensional cell spheres have a stronger pro-angiogenic paracrine potential.

[0041] Example 2 Establishment of a myocardial infarction model: Adult male C57 mice (weighing approximately 20–25 g) were anesthetized and mechanically ventilated via endotracheal intubation. A thoracotomy was performed at the left fourth intercostal space to expose the heart, and the left anterior descending artery (LAD) was ligated below the left atrial appendage to establish the myocardial infarction model. In the sham-operated group, only sutures were threaded without ligation. Immediately after ligation, whitening of the apex and anterior wall was observed as a sign of successful modeling. The chest wall was sutured, and postoperative analgesia and antibiotics were administered.

[0042] Cell therapy: After the model was established, the patients were randomly divided into three groups: MI group, MI+UCA-PSCs group, and MI+UCA-PSC sphere group (an additional Sham group was set up). On the 3rd day after ligation, 200 μL of UCA-PSC sphere suspension was injected subcutaneously into the left axilla, and two-dimensional UCA-PSCs with the same total number of UCA-PSC spheres were injected into the tail vein.

[0043] Cardiac function assessment: Echocardiography was performed at 2 and 4 weeks after treatment to measure indicators such as left ventricular ejection fraction (LVEF) and fractional shortening (FS).

[0044] The results are as follows Figure 3 As shown, compared with the MI group, both UCA-PSCs and UCA-PSC bulb therapy improved cardiac function indicators, with the UCA-PSC bulb group showing more significant improvements in LVEF / FS and left ventricular remodeling indicators. This suggests that cell bulb therapy has a superior protective effect on cardiac function recovery and left ventricular remodeling after myocardial infarction.

[0045] Mice were sacrificed 4 weeks after treatment. Hearts were perfused with PBS to remove blood and then fixed in 4% paraformaldehyde. They were then dehydrated in a conventional gradient and embedded in paraffin. Serial sections of the short axis of the heart were cut for immunofluorescence staining. After antigen retrieval, the sections were incubated with anti-CD31 primary antibody to label the vascular endothelium. Cell nuclei were counterstained with DAPI. High-power images of the whole heart, infarct area, and marginal area were obtained under a fluorescence microscope. The CD31 fluorescence intensity or vascular density was statistically analyzed using image analysis software.

[0046] The results are as follows Figure 4 As shown, compared with the MI group, UCA-PSCs treatment increased CD31 fluorescence signal in the infarct and marginal zones, with the increase being more significant in the UCA-PSC spheroid group. This suggests that spheroids have a stronger promoting effect on microvascular regeneration after myocardial infarction.

[0047] Heart samples from both treatment groups were subjected to transcriptome sequencing. Serum samples were subjected to proteome sequencing. Results are as follows: Figure 5 As shown: differential gene enrichment indicates a significant increase in key angiogenesis signaling pathways in the cell spheroid treatment group. Correlation analysis showed a significant negative correlation between S100A8 / S100A9 and sCD146, suggesting an association between sCD146 levels and the expression of inflammation-related molecules.

Claims

1. A peri-arterial stem cell sphere, characterized in that, The para-umbilical artery stem cell spheres are formed by three-dimensional culture of para-umbilical artery stem cells, with a diameter of 100-200 μm.

2. A method for preparing para-umbilical artery stem cell spheres, characterized in that, Includes the following steps: Para-umbilical artery stem cell culture: Para-umbilical artery stem cells were obtained by culturing them in complete culture medium at 30-40°C and 1-10% CO2. Preparation of para-arterial stem cell spheres: Adjust the concentration of the single-cell suspension to 1×10⁻⁶. 5 ~10×10 5 Cells per mL were seeded into ultra-low adsorption culture plates and cultured until the cells spontaneously aggregated to form cell spheres with a diameter of 100-200 μm.

3. A para-arterial stem cell sphere injection solution, characterized in that, It contains the para-umbilical artery stem cell sphere as described in claim 1.

4. A method for preparing a peri-arterial stem cell sphere injection solution, characterized in that, Includes the following steps: Para-umbilical artery stem cell culture: Para-umbilical artery stem cells were obtained by culturing them in complete culture medium at 30-40°C and 1-10% CO2. Preparation of para-arterial stem cell spheres: Adjust the concentration of the single-cell suspension to 1×10⁻⁶. 5 ~10×10 5 Cells / mL were seeded into ultra-low adsorption culture plates and cultured until the cells spontaneously aggregated to form cell spheres with a diameter of 100-200 μm. Preparation of para-umbilical artery stem cell sphere injection solution: Collect cell spheres, wash with physiological saline, and finally prepare para-umbilical artery stem cell sphere injection solution using 1-10% human serum albumin injection solution.

5. A method for detecting para-umbilical artery stem cell sphere injection solution, characterized in that, Cell viability and CD146 expression levels were detected.

6. The method for detecting the para-umbilical artery stem cell sphere injection solution according to claim 5, characterized in that, Cell viability of at least 90% and CD146 expression level significantly higher than that of two-dimensional cultured cells are considered qualified; otherwise, they are unqualified.

7. The use of the para-umbilical artery stem cell sphere of claim 1 or the para-umbilical artery stem cell sphere injection solution of claim 3 in the preparation of a medicament for treating ischemic cardiomyopathy.

8. The application according to claim 7, characterized in that, The ischemic cardiomyopathy includes acute myocardial infarction, post-myocardial infarction heart failure, or chronic ischemic cardiomyopathy.

9. The application according to claim 7, characterized in that, The dosage form of the drug includes injections.

10. A pharmaceutical composition, characterized in that, It comprises the para-umbilical artery stem cell sphere as described in claim 1 and a pharmaceutically acceptable carrier.

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