Method for identifying myocardial repair characteristics of umbilical cord mesenchymal stem cells

Abstract

The invention relates to a method for distinguishing myocardial repair characteristics of human umbilical cord mesenchymal stem cells through IL-33 expression level. Specifically, the invention provides a HucMSCs conditioned culture medium and a supernatant obtained by treating macrophages with the culture medium, the OGD damage resistance can be enhanced, and the HucMSCs conditioned culture medium with IL-33 high secretion has the strongest OGD damage resistance when treating the supernatant of the macrophages and has better myocardial repair capacity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for identifying the myocardial repair characteristics of umbilical cord mesenchymal stem cells. Background Art

[0002] Stem cell therapy is a promising new strategy for treating ischemic heart disease. The quality of stem cell preparations, dosing regimens, patient heterogeneity, etc. can affect the efficacy of stem cell therapy. To ensure the stability, safety and effectiveness of stem cell therapy, a quality management system for stem cell preparations needs to be established. Human umbilical cord mesenchymal stem cells (HucMSCs) have the advantages of sufficient source, youth, strong paracrine effects, etc., and are one of the ideal seed cell types for the development of stem cell therapy for ischemic heart disease. In recent years, studies have reported that HucMSCs from different donors have immunomodulatory heterogeneity, and there are subsets with different functions in HucMSCs from the same donor, but it has not been reported whether the heterogeneity of HucMSCs affects their myocardial repair effect. Therefore, developing HucMSCs therapy for ischemic heart disease requires answering whether its heterogeneity will lead to differences in myocardial repair efficacy. If it causes efficacy differences, how to control its heterogeneity?

[0003] We evaluated the repair effects of acute myocardial injection of different HucMSCs in a mouse myocardial infarction model and found that there were differences in their myocardial repair characteristics. To establish a method for differentiating the myocardial repair characteristics of HucMSCs, we discovered that IL-33 can be used as a marker for the myocardial repair characteristics of HucMSCs by revealing the molecular mechanisms leading to the generation of HucMSCs with high and low myocardial repair characteristics. This patent differentiates HucMSCs based on the high or low expression level of IL-33. The myocardial repair effects of HucMSCs with high and low IL-33 expression were compared in animal models and cell models to establish the correlation between high IL-33 expression levels of HucMSCs and high myocardial repair characteristics. In summary, our work reveals the heterogeneity of the myocardial repair characteristics of HucMSCs from different sources and for the first time establishes a method for differentiating the myocardial repair characteristics of human umbilical cord mesenchymal stem cells based on the IL-33 marker. Summary of the Invention

[0004] The first aspect of the present invention provides a method for identifying umbilical cord mesenchymal stem cells with high myocardial repair ability, including: detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL have higher myocardial repair ability compared to the umbilical cord mesenchymal stem cells with a content lower than 150 pg / mL.

[0005] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: The umbilical cord mesenchymal stem cells are cultured for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is obtained. The culture medium used is DMEM.

[0006] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: The umbilical cord mesenchymal stem cells are cultured for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours), the cells are collected, and then the collected cells are cultured for at least 10 hours (such as 24 hours) and then continue to be cultured for at least 20 hours, preferably 24 - 48 hours, to obtain the supernatant.

[0007] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows:

[0008] (1) The umbilical cord mesenchymal stem cells are cultured with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain the cells, and

[0009] (2) The cells obtained in (1) are cultured with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0010] In one or more embodiments, the concentration of the IL-33 is measured by the ELISA method.

[0011] In one or more embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0012] The present invention also provides a method for preparing umbilical cord mesenchymal stem cells with high myocardial repair ability, including:

[0013] (1) Detect the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells, and identify the umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL,

[0014] (2) Culture the umbilical cord mesenchymal stem cells identified in (1).

[0015] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: The umbilical cord mesenchymal stem cells are cultured with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is obtained. In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: (a) The umbilical cord mesenchymal stem cells are cultured with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain cells, and (b) The cells obtained in (a) are cultured with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0016] In one or more embodiments, step (2) includes: culturing umbilical cord mesenchymal stem cells in a medium to passage P5, preferably at a passage ratio of 1:3. In one or more embodiments, step (2) further includes: digesting and separating the cells. In one or more embodiments, the medium is a medium suitable for culturing umbilical cord mesenchymal stem cells, such as DMEM. Preferably, the medium contains serum or serum substitute.

[0017] In one or more embodiments, the concentration of IL-33 is measured by the ELISA method.

[0018] In one or more embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0019] In one or more embodiments, the method further includes the step of verifying the ability of umbilical cord mesenchymal stem cells to repair heart injury.

[0020] The present invention also provides the use of a reagent for specifically detecting IL-33 in the preparation of a reagent containing umbilical cord mesenchymal stem cells with high myocardial repair ability, and the reagent for specifically detecting IL-33 is used to detect the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells.

[0021] In one or more embodiments, the reagent for specifically detecting IL-33 is a reagent for detecting the expression level of IL-33. For example, an IL-33 specific binding molecule, including an antibody or its antigen-binding fragment.

[0022] In one or more embodiments, the reagent for specifically detecting IL-33 includes reagents used for detecting the content of IL-33 by one or more of Western blot, ELISA, and liquid chromatography-tandem mass spectrometry.

[0023] In one or more embodiments, the umbilical cord mesenchymal stem cells are prepared as follows:

[0024] (1) Detect the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells using a reagent specifically detecting IL-33, and identify umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL; and

[0025] (2) Culture the umbilical cord mesenchymal stem cells identified in (1).

[0026] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: Culture the umbilical cord mesenchymal stem cells with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant. In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: (a) Culture the umbilical cord mesenchymal stem cells with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain cells, and (b) Culture the cells obtained in (a) with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0027] In one or more embodiments, step (2) includes: Culturing the umbilical cord mesenchymal stem cells in a medium to passage P5, preferably at a passage ratio of 1:3. In one or more embodiments, step (2) further includes: Digesting and separating the cells. In one or more embodiments, the medium is a medium suitable for culturing umbilical cord mesenchymal stem cells, such as DMEM. Preferably, the medium contains serum or serum substitute.

[0028] In one or more embodiments, the concentration of IL-33 is measured by the ELISA method.

[0029] In one or more embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0030] In one or more embodiments, preparing umbilical cord mesenchymal stem cells further includes the step: (1.5) Verifying the ability of umbilical cord mesenchymal stem cells to repair heart injury.

[0031] The present invention also provides the use of umbilical cord mesenchymal stem cells in the preparation of a drug for repairing damaged myocardium or treating heart diseases, wherein the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells is higher than 150 pg / mL.

[0032] In one or more embodiments, the umbilical cord mesenchymal stem cells are prepared as follows:

[0033] (1) Detect the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells, and identify umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL,

[0034] (2) Culture the umbilical cord mesenchymal stem cells identified in (1).

[0035] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: culture the umbilical cord mesenchymal stem cells with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and obtain the culture supernatant. In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: (a) culture the umbilical cord mesenchymal stem cells with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours), obtain the cells, and (b) culture the cells obtained in (a) with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and obtain the culture supernatant.

[0036] In one or more embodiments, step (2) includes: culturing the umbilical cord mesenchymal stem cells in a medium to passage P5, preferably at a passage ratio of 1:3. In one or more embodiments, step (2) further includes: digesting and separating the cells. In one or more embodiments, the medium is a medium suitable for culturing umbilical cord mesenchymal stem cells, such as DMEM. Preferably, the medium contains serum or serum substitute.

[0037] In one or more embodiments, the concentration of the IL-33 is measured by the ELISA method.

[0038] In one or more embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0039] In one or more embodiments, the myocardial injury is myocardial injury caused by ischemic heart disease, and / or oxygen-glucose deprivation (OGD) myocardial injury.

[0040] In one or more embodiments, the heart diseases include: myocardial infarction, ischemic heart disease, myocardial injury caused by ischemic heart disease, or heart failure.

[0041] In one or more embodiments, the preparation of umbilical cord mesenchymal stem cells further includes the step: (1.5) verifying the ability of the umbilical cord mesenchymal stem cells to repair heart injury.

[0042] The present invention also provides a pharmaceutical composition, comprising umbilical cord mesenchymal stem cells and pharmaceutically acceptable excipients, wherein the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells is higher than 150 pg / mL.

[0043] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: The umbilical cord mesenchymal stem cells are cultured with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is obtained. In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: (1) The umbilical cord mesenchymal stem cells are cultured with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain cells, and (2) The cells obtained in (1) are cultured with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0044] In one or more embodiments, the umbilical cord mesenchymal stem cells are prepared as follows:

[0045] (1) Detect the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells, and identify the umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL.

[0046] (2) Culture the umbilical cord mesenchymal stem cells identified in (1).

[0047] In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: The umbilical cord mesenchymal stem cells are cultured with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is obtained. In one or more embodiments, the culture supernatant of the umbilical cord mesenchymal stem cells described in step (1) is prepared as follows: (a) The umbilical cord mesenchymal stem cells are cultured with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain cells, and (b) The cells obtained in (a) are cultured with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0048] In one or more embodiments, step (2) includes: Culturing the umbilical cord mesenchymal stem cells to passage P5 in a culture medium, preferably at a passage ratio of 1:3. In one or more embodiments, step (2) further includes: Digesting and separating the cells. In one or more embodiments, the culture medium is a culture medium suitable for culturing umbilical cord mesenchymal stem cells, such as DMEM. Preferably, the culture medium contains serum or serum substitute.

[0049] In one or more embodiments, the concentration of the IL-33 is measured by the ELISA method.

[0050] In one or more embodiments, preparing the umbilical cord mesenchymal stem cells further includes the step of: (1.5) Verifying the ability of the umbilical cord mesenchymal stem cells to repair heart injury.

[0051] In one or more embodiments, the pharmaceutically acceptable excipient is physiological saline.

[0052] In one or more embodiments, the drug is used for treating heart diseases, preferably including: myocardial infarction, ischemic heart disease, myocardial injury caused by ischemic heart disease or heart failure.

[0053] The present invention also provides a method for treating and repairing myocardium or treating heart diseases, comprising the step of administering the pharmaceutical composition described herein to a patient.

[0054] In one or more embodiments, the heart diseases include: myocardial infarction, ischemic heart disease, myocardial injury caused by ischemic heart disease or heart failure.

[0055] The present invention also provides the use of a reagent for specifically detecting IL-33 in the preparation of a kit for identifying the myocardial injury repair ability of umbilical cord mesenchymal stem cells. The myocardial injury repair ability includes myocardial repair effect or efficacy.

[0056] In one or more embodiments, the reagent for specifically detecting IL-33 is a reagent for specifically detecting the expression level of IL-33, such as an IL-33 specific binding molecule, including an antibody or an antigen-binding fragment thereof.

[0057] In one or more embodiments, the reagent for specifically detecting IL-33 includes the reagents used for detecting the content of IL-33 by using one or more of Western blot, ELISA and liquid chromatography-tandem mass spectrometry.

[0058] In one or more embodiments, identifying the myocardial injury repair ability of umbilical cord mesenchymal stem cells includes: detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells. If the content of IL-33 is higher than 150 pg / mL (such as 150 - 200 pg / mL, preferably 160 - 190 pg / mL), then the umbilical cord mesenchymal stem cells have high myocardial repair ability.

[0059] In one or more embodiments, the culture supernatant of umbilical cord mesenchymal stem cells is prepared as follows: culturing umbilical cord mesenchymal stem cells with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and obtaining the culture supernatant.

[0060] In one or more embodiments, the culture supernatant of umbilical cord mesenchymal stem cells is prepared as follows:

[0061] (1) Culturing umbilical cord mesenchymal stem cells with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours), obtaining cells, and

[0062] (2) Culture the cells obtained in (1) in serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant.

[0063] In one or more embodiments, between steps (1) and (2), there is also a step of washing the cells with a buffer.

[0064] In one or more embodiments, the cardiac muscle is cardiac muscle of a mammal (preferably human).

[0065] In one or more embodiments, the myocardial injury is myocardial injury caused by ischemic heart disease.

[0066] The present invention also provides a kit for identifying the myocardial repair ability of umbilical cord mesenchymal stem cells, comprising a reagent for specifically detecting IL-33 and an umbilical cord mesenchymal stem cell culture medium.

[0067] In one or more embodiments, the kit further comprises one or more selected from the following: washing buffer, serum, serum substitute.

[0068] In one or more embodiments, the reagent for specifically detecting IL-33 includes reagents used for detecting the content of IL-33 by one or more of Western blot, ELISA, and liquid chromatography - tandem mass spectrometry.

[0069] In one or more embodiments, the reagent for specifically detecting IL-33 is a reagent for specifically detecting the expression level of IL-33. For example, an IL-33 specific binding molecule, including an antibody or its antigen-binding fragment.

[0070] The present invention also provides a conditioned medium, which is prepared by the following method:

[0071] (1) Identify umbilical cord mesenchymal stem cells with high myocardial repair ability as described in any embodiment of the first aspect of the present application,

[0072] (2) Culture umbilical cord mesenchymal stem cells in DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 24 - 48 hours) to obtain cells, and

[0073] (3) Culture the cells obtained in (1) in serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is the conditioned medium.

[0074] In one or more embodiments, the conditioned medium is a macrophage-conditioned medium.

[0075] In one or more embodiments, the content of IL-33 in the conditioned medium is higher than 150 pg / mL, such as 150 - 200 pg / mL, preferably 160 - 190 pg / mL.

[0076] In one or more embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0077] In one or more embodiments, the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells capable of differentiating into adipocytes, osteoblasts or chondrocytes.

[0078] In one or more embodiments, between steps (2) and (3), there is also a step of washing the cells with a buffer.

[0079] In one or more embodiments, the concentration of the IL-33 is measured by the ELISA method.

[0080] The present invention also provides a method for inducing macrophages to polarize into a reparative type, including culturing macrophages with the conditioned medium described in any one of the embodiments herein.

[0081] In one or more embodiments, the method includes: culturing macrophages with the conditioned medium described in any one of the embodiments herein for at least 10 hours, preferably at least 24 hours, more preferably 24 - 30 hours.

[0082] The third aspect of the present invention provides a composition having the ability to resist myocardial injury, and the composition contains the culture supernatant obtained by culturing macrophages with the conditioned medium described in any one of the embodiments herein. In one or more embodiments, the culturing conditions include one or more of the following: 30 - 45 °C (preferably 37 °C), 5% CO 2 , culturing for at least 10 hours (preferably 24 hours).

[0083] The present invention also provides a method for preparing the composition described in the third aspect, including the following steps: culturing macrophages with the conditioned medium described in any one of the embodiments herein to obtain the supernatant. In one or more embodiments, the culturing conditions include one or more of the following: 30 - 45 °C (preferably 37 °C), 5% CO 2 , culturing for at least 10 hours (preferably 24 hours). BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 : Identification of Markers, Morphology and Differentiation Ability of Passage 5 HucMSCs

[0085] (A) Identification of HucMSCs markers by flow cytometry (positive markers: CD73, CD90, and CD105; negative markers: CD11b, CD19, CD34, CD45, and HLA-DR)

[0086] (B) Morphology of HucMSCs

[0087] (C) Identification of the ability of HucMSCs to differentiate into adipocytes

[0088] (D) Identification of the ability of HucMSCs to differentiate into osteoblasts

[0089] (E) Identification of the ability of HucMSCs to differentiate into chondrocytes

[0090] Figure 2 : Regulatory effect of HucMSCs conditioned medium (HucMSCs-CdM) on macrophages

[0091] (A-D) Effects of secreted components of HucMSCs on the viability (A), morphology (B), and number (C) of primary macrophages, n = 4.

[0092] (D) Secreted components of HucMSCs induce macrophage polarization towards the reparative type, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significant difference.

[0093] Figure 3 : IL-33 secretion levels of HucMSCs-CdM from different umbilical cord sources

[0094] (A) hIL-33 content in H-MSCs-CdM and L-MSCs-CdM, n = 4. **p < 0.01.

[0095] Figure 4 : Effect of treating macrophages with HucMSCs-CdM on protecting cardiomyocytes against OGD injury

[0096] (A-B) Treatment of macrophages with HucMSCs Representative diagram (A) and statistical chart (B) comparing the anti-OGD injury effects of secreted components on cardiomyocytes, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significant difference.

[0097] Figure 5 : There are differences in the protective effects of transplanted HucMSCs from different sources on cardiac function in infarcted hearts

[0098] (A) Schematic diagram of the myocardial infarction model and echocardiogram detection mode

[0099] (B) Effects of transplanted HucMSCs on cardiac function indexes (LVEF, LVFS), n = 6 - 9. ***p < 0.001 Detailed implementation manners

[0100] Unless otherwise defined, the implementation of the present invention will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of those skilled in the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Animal Cell Culture (edited by R.I. Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987 edition and its regularly updated versions); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0101] The inventors first revealed that by detecting the expression level of the IL-33 gene in the conditioned medium of HucMSCs, the strength of myocardial repair characteristics can be significantly regulated, and further the differences in myocardial repair efficacy can be distinguished.

[0102] The first aspect of the present invention provides a method for identifying umbilical cord mesenchymal stem cells with high myocardial repair ability, including: detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells, and if the content is higher than 150 pg / mL, then the umbilical cord mesenchymal stem cells have high myocardial repair ability.

[0103] As used herein, "culture supernatant" refers to the cell-free liquid obtained during the process of culturing cells. Usually, the supernatant can be obtained after culturing cells with a suitable medium for a period of time. The methods for obtaining the supernatant from the culture composition are well-known in the art, such as digestion, filtration, centrifugation, etc.

[0104] In one or more embodiments, the culture supernatant of umbilical cord mesenchymal stem cells is prepared as follows: The umbilical cord mesenchymal stem cells are cultured with DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant. Specifically, the culture supernatant of umbilical cord mesenchymal stem cells is prepared as follows: (1) The umbilical cord mesenchymal stem cells are cultured with DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours) to obtain cells, and (2) The cells obtained in (1) are cultured with serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours) to obtain the culture supernatant. A step of washing the cells with a buffer may be included between the above steps (1) and (2). The buffers mainly include phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, Tris, etc. In any embodiment herein, the buffer includes DPBS.

[0105] In one or more embodiments, the present invention also relates to the culture supernatant of macrophages, which is prepared as follows: The macrophages are cultured with the conditioned medium described in any embodiment herein to obtain the supernatant.

[0106] In this article, "cardiomyocyte reparative", "cardiomyocyte repair ability", "cardiomyocyte injury reparative", and "cardiomyocyte injury repair ability" can be used interchangeably, and all represent the ability to repair damaged cardiomyocytes to restore or partially restore their morphology, integrity, or function. "High cardiomyocyte reparative" refers to the ability of the umbilical cord mesenchymal stem cell experimental group to significantly improve cardiac function indexes (such as LVEF, LVFS) compared with the control group. The control group can be normal saline or umbilical cord mesenchymal stem cells with an IL-33 content lower than 150 pg / ml in the culture supernatant. In this article, a significant increase means that: compared with the control sample, the increase amplitude of the cardiac function indexes (such as LVEF, LVFS) is ≥10%, preferably ≥20%, preferably ≥50%, more preferably ≥80%, and most preferably ≥100%.

[0107] In this article, exemplary umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0108] The present invention includes a method for detecting the concentration of IL-33 in the culture supernatant. Any method commonly used in the art for the specific quantitative detection of IL-33 can be used, such as Western blot, ELISA, liquid chromatography-tandem mass spectrometry, etc. There are no particular limitations on these methods in this article. At the same time, the reagents used in the above methods are also well-known and easily obtainable by those skilled in the art, which include IL-33 specific reagents and other required reagents, and these reagents are also included within the scope of the present invention. IL-33 specific reagents are usually IL-33 specific binding molecules (including antibodies or their antigen-binding fragments). Antibodies can be easily obtained by those skilled in the art based on the structure and sequence of IL-33 and its coding sequence, or can be commercially available. In one or more embodiments, the concentration of the IL-33 is measured by an ELISA method, such as the Human IL-33 ELISA Kit from Beyotime.

[0109] The above method for identifying umbilical cord mesenchymal stem cells with high myocardial reparability may further include the step of verifying the cardiac injury repair ability of the umbilical cord mesenchymal stem cells. Such a verification step can generally be achieved by measuring the expression levels of protein / molecular markers, cell experiments, tissue experiments, in vivo experiments, etc. For example, it can be verified by administering umbilical cord mesenchymal stem cells to mice with myocardial infarction or myocardial infarction model mice, and the administration is, for example, injection. By comparing with a control (such as normal saline and known umbilical cord mesenchymal stem cells with low myocardial reparability), the cardiac injury repair ability of the umbilical cord mesenchymal stem cells can be determined.

[0110] The present invention also provides a method for preparing umbilical cord mesenchymal stem cells with high myocardial reparability, including: identifying umbilical cord mesenchymal stem cells with high myocardial reparability as described above, and culturing the identified umbilical cord mesenchymal stem cells. For example, after obtaining umbilical cord mesenchymal stem cells, a part of them can be taken and cultured to obtain the supernatant for identifying myocardial reparability using the method described herein. When it is identified that the content of IL-33 in the culture supernatant is higher than 150 pg / mL or higher, the obtained umbilical cord mesenchymal stem cells are cultured to obtain umbilical cord mesenchymal stem cells with high myocardial reparability.

[0111] After identifying umbilical cord mesenchymal stem cells with high myocardial reparability, there are no particular limitations on the method for culturing them. Therefore, any culture method for mesenchymal stem cells well-known in the art can be used in the present invention. Exemplary culture methods include: culturing umbilical cord mesenchymal stem cells in DMEM with or without serum or serum substitute at a passage ratio of 1:3 to passage P5, digesting the cells (such as trypsin treatment), and separating the cells (such as centrifugation).

[0112] Another embodiment of the present invention is a cell culture containing the umbilical cord mesenchymal stem cells with high myocardial reparability.

[0113] Some other embodiments of the present invention are cryopreparations of any of the umbilical cord mesenchymal stem cells or cell cultures described herein.

[0114] The present invention also provides a method for treating and preventing myocardial injury or related symptoms in a subject in need thereof, the method comprising the step of administering to the subject in need thereof a therapeutically effective amount of the umbilical cord mesenchymal stem cells, cell cultures or pharmaceutical compositions described herein. As used herein, myocardial injury particularly includes myocardial injury caused by ischemic heart disease. Symptoms related to myocardial injury mainly refer to heart diseases related to myocardial injury, including but not limited to myocardial infarction, ischemic heart disease, and heart failure caused by ischemic heart disease. The oxygen-glucose deprivation (OGD) model is a well-recognized myocardial injury model in the art. In certain embodiments, the preferred subjects are mammals or birds, and most preferably humans. In certain embodiments, the method for treating and preventing diseases includes combination therapy with one or more other drugs.

[0115] As used herein, the phrase "therapeutically effective amount" means an amount sufficient to cause a clinically significant improvement in the subject, or to delay or minimize or alleviate one or more symptoms related to the disease, or to cause a physiologically desired beneficial change in the subject. The term "treatment" refers to slowing down, alleviating, improving or reducing at least one symptom of a disease, or reversing its onset after the disease has occurred. The term "prevention" refers to acting before the onset of the disease to prevent the formation of the disease or to reduce the severity of the disease or to delay its development. As used in this application, the term "subject" refers to an animal with an immune system, such as birds and mammals.

[0116] Some other embodiments of the present invention are pharmaceutical compositions, wherein the pharmaceutical compositions comprise a therapeutically effective amount of any of the umbilical cord mesenchymal stem cells or cell cultures described herein and pharmaceutically acceptable excipients.

[0117] The pharmaceutical composition may comprise any number of umbilical cord mesenchymal stem cells. For example, one unit dose of umbilical cord mesenchymal stem cells may comprise, at least approximately, or not more than 1×10 5 、5×10 5 、1×10 6 、5×10 6 、1×10 7 、5×10 7 、1×10 8 、5×10 8 、1×10 9 、5×10 9 、1×10 10 、5×10 10 、1×10 11 or more umbilical cord mesenchymal stem cells.

[0118] The pharmaceutical compositions disclosed herein comprise cell populations that contain 50% or more viable cells (i.e., at least 50% of the cells in the cell population are functional or alive). Preferably, at least 60% of the cells in the cell population are alive. More preferably, in the pharmaceutical composition, at least 70%, 80%, 90%, 95% or 99% of the cells in the cell population are alive.

[0119] The phrase "pharmaceutically acceptable" means that the molecular entities and compositions are physiologically tolerable and generally do not produce allergic or similar reactions, such as stomach discomfort, dizziness, etc. after administration to humans. And are approved by a government regulatory agency, or listed in national pharmacopoeias or other recognized pharmacopoeias for animals, and more particularly for humans. "Excipients" refer to diluents, adjuvants, excipients or carriers approved for the medicament. Such pharmaceutical carriers can be sterile liquids, such as aqueous saline solutions and oils, including petroleum, animal oils, vegetable oils or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, a physiological saline solution is the preferred carrier. Aqueous saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, especially for injectable liquids. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, ethylene glycol, water, ethanol, etc. If desired, these compositions also contain trace amounts of wetting agents, emulsifying agents and / or pH buffering agents.

[0120] These compositions can be in the following forms: solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, cachets, lozenges, troches, dispersions, suppositories, ointments, cataplasms (mud packs), pastes, dressings, creams, plasters, patches, aerosols, gels. Liquid dosage forms are suitable for parenteral administration to a patient, and sterilized solid preparations (such as crystalline or amorphous solids) are reconstituted into liquid dosage forms for parenteral administration to a patient. Such compositions should contain a therapeutically effective amount of the said compound (preferably in purified form) and a suitable amount of carrier in order to provide a suitable dosage form for administration to the patient.

[0121] Pharmaceutical compositions suitable for oral administration can be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids) or emulsions. Tablets or hard gelatin capsules can include lactose, starch or its derivatives, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules can include vegetable oils, waxes, fats, semi-solids, or liquid polyols. Solutions and syrups can contain water, polyols, and sugars. Active agents designed for oral administration can be coated with or mixed with substances that delay the disintegration and / or absorption of the active agent in the gastrointestinal tract. Thus, sustained release of the active agent over a number of hours can be achieved, and if desired, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions formulated for oral administration facilitate the release of the active agent at a specific gastrointestinal location. Pharmaceutical compositions for oral administration can be formulated to assist the release of the active agent at a specific gastrointestinal location (due to special pH or enzyme conditions).

[0122] Pharmaceutical compositions suitable for nasal and pulmonary administration can contain solid carriers such as powders that can be rapidly administered through the nose. Pharmaceutical compositions for nasal mucosal administration can include liquid carriers, such as nasal sprays or nasal drops. Additionally, direct inhalation into the lungs can be achieved by taking a deep breath or using a blowpipe. These compositions can contain aqueous or oily solutions of the active ingredient. Compositions for administration by inhalation can be packaged in specially adapted devices, including but not limited to pressurized aerosols, nebulizers, or insufflators, and such devices can be manufactured so as to provide a predetermined dose of the active ingredient.

[0123] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or suspensions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the composition substantially isotonic with the blood of a predetermined recipient. Other ingredients that can be present in such compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. Compositions suitable for parenteral administration can be present in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions. In such cases, only a sterile liquid carrier need be rapidly added immediately prior to use. Temporary injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Suitable carriers for providing the parenteral dosage forms of the present invention are well known to those skilled in the art. Examples include: USP Water for Injection; aqueous carriers such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0124] The selection of the effective dose will be determined by those skilled in the art based on the consideration of several factors well-known to those of ordinary skill in the art. These factors include the specific form of the inhibitor and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be established in the conventional development procedures, and usually these procedures are required to obtain the regulatory approval of the drug compound. Other factors related to the dose include the condition or disease to be treated, or the benefit to be obtained for normal people, the weight of the patient, the route of administration, whether the administration is acute or chronic, concomitant administration, and other well-known factors that affect the efficiency of the administered agent. Therefore, the precise dose should be determined according to the judgment of the physician and the specific situation of each patient.

[0125] The preparation of the pharmaceutical composition described herein may also require reagents for specifically detecting IL-33, and the reagents for specifically detecting IL-33 are used to detect the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells. Umbilical cord mesenchymal stem cells with a content of IL-33 higher than 150 pg / mL can be used for the preparation of the pharmaceutical composition.

[0126] The present invention also provides a kit, which comprises the pharmaceutical composition described herein and reagents for specifically detecting IL-33.

[0127] The umbilical cord mesenchymal stem cells disclosed herein can be used to produce immunosuppressive conditioned medium, that is, the medium contains one or more biomolecules secreted or excreted by the stem cells. The content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells is higher than 150 pg / mL, such as 150 - 200 pg / mL, preferably 160 - 190 pg / mL. In various embodiments, the conditioned medium includes the medium in which the umbilical cord mesenchymal stem cells have grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium includes the medium in which the umbilical cord mesenchymal stem cells have grown to a confluence of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or reached 100%. Such conditioned medium can be used to support the culture of a single umbilical cord mesenchymal stem cell population or the culture of other types of stem cells.

[0128] In one embodiment, the conditioned medium herein is prepared as follows: (1) Identify umbilical cord mesenchymal stem cells with high myocardial reparability as described in any embodiment of the present application; (2) Culture the umbilical cord mesenchymal stem cells in DMEM containing serum or serum substitute for at least 10 hours (preferably at least 48 hours, such as 48 - 72 hours), obtain the cells, and (3) Culture the cells obtained in (2) in serum-free DMEM for at least 10 hours (preferably at least 20 hours, such as 24 - 48 hours), and the culture supernatant is the conditioned medium. In one or more embodiments, the content of IL-33 in the conditioned medium is higher than 150 pg / mL, such as 150 - 200 pg / mL, preferably 160 - 190 pg / mL.

[0129] Therefore, in one embodiment, the present invention provides a composition comprising a culture medium and / or other umbilical cord mesenchymal stem cell derivatives, wherein the culture supernatant of the umbilical cord mesenchymal stem cells contains a high content of IL-33.

[0130] In some embodiments herein, the conditioned medium is macrophage-conditioned medium. The inventors found that the conditioned medium derived from umbilical cord mesenchymal stem cells with high myocardial reparability as described herein can induce an increase in the number of reparative macrophages; the supernatant obtained after culturing macrophages with this conditioned medium has high myocardial injury repair ability. The present invention provides a method for culturing macrophages using the umbilical cord mesenchymal stem cells described herein as feeder cells. The present invention provides a co-culture of umbilical cord mesenchymal stem cell macrophages as described herein.

[0131] The present invention also provides a kit for identifying the myocardial reparability of umbilical cord mesenchymal stem cells, comprising a reagent for specifically detecting IL-33 and an umbilical cord mesenchymal stem cell culture medium. The kit may further comprise one or more selected from the following: washing buffer, serum, serum substitute. Herein, the serum is selected from human AB serum, the subject's autologous serum or animal-derived serum. In one or more embodiments, the concentration of the serum is 1 - 10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of the serum is 1.5 - 9.5%, 2.5 - 8.5%, 3.5 - 7.5%, 4.5 - 6.5%.

[0132] The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0133] Examples

[0134] Example 1: Detection of the level of IL-33 secreted by HucMSCs and its regulatory ability on macrophages

[0135] 1.1 Collection of conditioned medium of HucMSCs and detection of IL-33 content

[0136] HucMSCs were cultured in DMEM medium containing 5% serum substitute and amplified to passage 5 at a passage ratio of 1:3. Passage 5 HucMSCs (provided by the GMP laboratory of the Stem Cell Base of the Dongfang Hospital Affiliated to Tongji University. The original cell isolation method was as follows: The neonatal umbilical cord was soaked in PBS, the artery and vein were discarded, and the tissue was mechanically minced into 1-2 mm 3 tissue blocks with scissors. The tissue blocks were digested with trypsin and dispase, etc., and the digestion was terminated. The tissue blocks and cell suspension were resuspended with DMEM + 5% serum substitute medium and plated in a 10 cm culture dish. They were cultured in an incubator at 37 °C and 5% CO 2 . Fresh medium was changed every other day.). 2.5×10 5 cells were taken into a 6-well plate and cultured overnight with improved Eagle's basal medium (DMEM, ThermoFisher Scientific, C11330500CP) containing 5% serum substitute (Helios Bioscience, UltraGRO serum substitute) (2 mL of the culture medium, cultured in an environment of 37 °C and 5% CO 2 ) to allow them to adhere. The next day, the culture medium was discarded, washed once with Dulbecco's phosphate buffered saline (DPBS, ThermoFisher Scientific, C14190500CP), and 2 mL of serum-free DMEM was added and cultured for 48 hours. The cell culture medium was collected, centrifuged at 1000 rpm for 3 minutes, and the supernatant was collected for use. The IL-33 content in the conditioned medium of HucMSCs was detected according to the instructions of the Human IL-33 ELISA Kit (Human IL-33 ELISA Kit, Beyotime, PI631).

[0137] 1.2 Isolation, culture and viability determination of macrophages

[0138] Prepare a thioglycollate medium solution with a concentration of 30 g / L using distilled water. Three days before isolating mouse peritoneal macrophages, inject 1 ml of the thioglycollate medium solution into the peritoneal cavity. Sacrifice the mouse by cervical dislocation, inject 10 ml of RPMI-1640 medium into the peritoneal cavity, massage the abdomen for 3 min, aspirate the cell suspension, filter it through a 40-μm filter and lyse the red blood cells. Resuspend the cells in DMEM medium containing 10% fetal bovine serum (FBS, Gibico, 10100147) and 1% antibiotics (PS, Gibico, 15140122). Count the cells using a cell counting chamber. After the cells adhere for 4 hours, change the medium to remove non-adherent cells and continue culturing. Culture the macrophages in DMEM medium containing 10% fetal bovine serum (FBS, Gibico, 10100147) and 1% antibiotics (PS, Gibico, 15140122) for 24 hours, and then treat them with the supernatant of HucMSCs obtained in 1.1 or serum-free DMEM for 24 hours. Replace the supernatant of HucMSCs containing 10% CCK8 reagent and continue culturing for 6 hours. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm.

[0139] 1.3 Isolation of cardiomyocytes and establishment and evaluation of the oxygen-glucose deprivation (OGD) model

[0140] Isolate adult SD rat cardiomyocytes using the Langendorff method. Prepare cardiomyocyte-depleted blood (take 500 mL of ddH 2 O, add 125 mM NaCl, 8 mM KCl, 1.2 mM KH 2 PO 4 4, 1.25 mM MgSO 4 4, 20 mM HEPES, 6.25 mM NaHCO 3 3, 5 mM sodium lactate, 1.2 mM CaCl 2 2, and adjust the pH to 6.6). Add cardiomyocyte-depleted blood or medium (HucMSCs conditioned medium or the medium after treating macrophages with HucMSCs conditioned medium), and transfer the culture dish to an incubator at 37 °C with an oxygen concentration of 0.1% for 1 hour. Take out the cells and observe the ratio of rod-shaped cardiomyocytes to round cardiomyocytes. The ratio of rod-shaped to round cardiomyocytes in the OGD injury group is approximately 1:1. Collect the supernatant and measure the absorbance of the supernatant at 490 nm for lactate dehydrogenase (LDH) to calculate the cytotoxicity.

[0141] 1.4 Statistics

[0142] All statistical data are expressed as mean ± SEM. Unpaired Student's t-test or one-way analysis of variance (ANOVA) is used to analyze the significance. Statistical analysis is performed using Graphpad Prism 8 software, and p < 0.05 indicates a significant difference.

[0143] Example 2: Comparison of the Efficacy of HucMSCs Transplanted by Myocardial Injection in Treating Myocardial Infarction in C57BL / 6 Mice

[0144] 2.1 Preparation of Transplanted Cells

[0145] HucMSCs were cultured in DMEM medium containing 5% serum substitute (Helios Bioscience, UltraGRO serum substitute) and amplified to passage 5 at a passage ratio of 1:3. After digestion with 0.25% trypsin, after terminating the digestion, centrifuged at 1000 rpm for 3 minutes, discarded the supernatant, and the cell pellet was resuspended with 0.9% normal saline, and the density was adjusted to 5×10 5 cells / 20 μL.

[0146] 2.2 Preparation of C57BL / 6 Myocardial Infarction Mouse Model and Cell Transplantation

[0147] Male C57BL / 6 mice at 10 - 12 weeks old were anesthetized and thoracotomized. The left anterior descending coronary artery was ligated 2 - 4 mm below the lower edge of the auricle using 8-0 nylon thread to make the model. After ligation, 20 μL of normal saline or HucMSCs (dose 5×10 5 cells / 20 μL) were injected at three points at the edge of the myocardial infarction. Finally, the ribs and skin were sutured. After the mice woke up, the ventilator was removed and the mice were placed in the cage.

[0148] 2.3 Cardiac Function Detection

[0149] Transthoracic echocardiography was used to detect the cardiac function of mice at 2, 7, and 28 days after myocardial infarction, and various cardiac function indexes such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were synchronously recorded. The analysis data of cardiac function on the 2nd day after myocardial infarction by transthoracic echocardiography was used as an index to judge whether the severity of acute myocardial infarction prepared by surgical operation was consistent.

[0150] 2.4 Statistics

[0151] All statistical data were expressed as mean ± SEM. The data of transthoracic echocardiography were analyzed by two-way ANOVA and Tukey's method for multiple comparisons. Graphpad Prism 8 software was used for statistical analysis, and p < 0.05 was considered to have significant differences.

[0152] Experimental Results

[0153] I. There are differences in the ability of HucMSCs from different host sources to regulate macrophages and the level of secreted IL-33

[0154] Obtain HucMSCs from different umbilical cord sources ( Figure 1)。Collect the conditioned medium of HucMSCs, treat peritoneal macrophages for 24 hours, and perform cell counting and CCK8 assay (Dojindo Laboratories, CK04-1000T). It was found that there were differences in the regulatory effects of conditioned media from HucMSCs of different host origins on the increase in macrophage number and enhancement of viability ( Figure 2 )。A total of 8 umbilical cord-derived HucMSCs (coded as No. 1-8) were obtained. Among them, CdM-2 and CdM-5 were the conditioned media of No. 2 and No. 5 HucMSCs-CdM respectively, and there were significant differences in their macrophage activity regulation. ELISA method (Beyotime, PI631) was used to detect the secretion level of IL-33 in the conditioned medium of HucMSCs. It was found that there were significant differences in the secretion ability of HucMSCs of different host origins, and the HucMSCs with stronger promotion of macrophage proliferation had higher IL-33 secretion ability (CdM > 150 pg / ml) ( Figure 3 , and the figure shows the combined results of all highly regulated and lowly regulated HucMSCs). Further collect the supernatant of the conditioned medium of HucMSCs in 1.1 to treat macrophages (37 °C, 5% CO 2 The incubator was used to treat macrophages for 24 h), and detect its ability to resist OGD injury of cardiomyocytes. It was found that the ability of the conditioned medium of HucMSCs to resist OGD injury was enhanced after treating macrophages, and the supernatant of the conditioned medium of HucMSCs with high IL-33 secretion had the strongest ability to resist OGD injury when treating macrophages ( Figure 4 )。

[0155] II. Highly regulated HucMSCs have better myocardial repair effects

[0156] During the acute phase of myocardial infarction in mice, 20 μL / 500,000 highly regulated or lowly regulated HucMSCs were injected into the myocardium (highly regulated cells had a more significant effect on promoting macrophage proliferation, and lowly regulated cells had a weaker effect on promoting macrophage proliferation; among them, the CCK8 index of macrophages was greater than or equal to 1.2 times and the cell number was greater than or equal to 1.5 times that of the DMEM control group were highly regulated), and 20 μL of normal saline was used as a control. Echocardiography was used to detect the cardiac function indexes LVEF and LVFS on the second day after myocardial infarction, and there were no significant differences among groups, indicating the consistency of the surgery. The cardiac function was detected on the 7th and 28th days after myocardial infarction. The HucMSCs transplantation group significantly improved the cardiac function indexes LVEF and LVFS compared with the normal saline control group, and the highly regulated HucMSCs group had a significantly better improvement effect than the lowly regulated HucMSCs group. It was shown that highly regulated HucMSCs protected cardiac function better than lowly regulated HucMSCs.

Claims

1. A method for identifying umbilical cord mesenchymal stem cells with high myocardial repair ability, comprising: detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells.

2. The method according to claim 1, wherein, umbilical cord mesenchymal stem cells with an IL-33 content in the supernatant higher than 150 pg / mL have higher myocardial repair ability compared to umbilical cord mesenchymal stem cells with an IL-33 content lower than 150 pg / mL, preferably, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: culturing the umbilical cord mesenchymal stem cells for at least 10 hours, and obtaining the culture supernatant, more preferably, the culture supernatant of the umbilical cord mesenchymal stem cells is prepared as follows: (1) culturing umbilical cord mesenchymal stem cells with DMEM containing serum or serum substitute for at least 10 hours to obtain cells, and (2) culturing the cells obtained in (1) with serum-free DMEM for at least 10 hours to obtain the culture supernatant.

3. Use of a reagent for specifically detecting IL-33 in the preparation of a reagent containing umbilical cord mesenchymal stem cells with high myocardial repair ability, wherein the reagent for specifically detecting IL-33 is used to detect the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells, preferably, the reagent for specifically detecting IL-33 is a reagent for specifically detecting the expression level of IL-33.

4. Use of umbilical cord mesenchymal stem cells in the preparation of a drug for repairing damaged myocardium or treating heart diseases, wherein the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells is higher than 150 pg / mL, preferably, the umbilical cord mesenchymal stem cells are prepared as follows: (1) detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells, identifying umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL, and (2) culturing the umbilical cord mesenchymal stem cells identified in (1), more preferably, the myocardial injury is myocardial injury caused by ischemic heart disease and / or oxygen-glucose deprivation (OGD) myocardial injury; the heart diseases include: myocardial infarction, ischemic heart disease, myocardial injury caused by ischemic heart disease or heart failure.

5. A pharmaceutical composition comprising umbilical cord mesenchymal stem cells and a pharmaceutically acceptable excipient, wherein the content of IL-33 in the culture supernatant of the umbilical cord mesenchymal stem cells is higher than 150 pg / mL, preferably, the umbilical cord mesenchymal stem cells are prepared as follows: (1) detecting the content of IL-33 in the culture supernatant of umbilical cord mesenchymal stem cells, identifying umbilical cord mesenchymal stem cells with a content higher than 150 pg / mL, and (2) culturing the umbilical cord mesenchymal stem cells identified in (1).

6. Use of a reagent for specifically detecting IL-33 in the preparation of a kit for differentiating the myocardial injury repair ability of umbilical cord mesenchymal stem cells.

7. A kit for identifying the myocardial repair ability of umbilical cord mesenchymal stem cells, comprising a reagent for specifically detecting IL-33 and a culture medium for umbilical cord mesenchymal stem cells.

8. A conditioned medium is prepared by the following method: (1) identifying umbilical cord mesenchymal stem cells with high myocardial repair ability as described in claim 1, (2) Culture umbilical cord mesenchymal stem cells with DMEM containing serum or serum substitute to obtain cells, and (3) Culture the cells obtained in (1) with serum-free DMEM, and the culture supernatant is the conditioned medium described above.

9. A composition having the ability to resist myocardial injury, the composition comprising the culture supernatant obtained by culturing macrophages with the conditioned medium described in claim 8.

10. A method for preparing the composition as described in claim 9, comprising the following steps: culture macrophages with the conditioned medium described in claim 8 to obtain the supernatant.

Images