TWI785479B - Pharmaceutical preparations of amniotic fluid-derived cardiomyocytes and uses thereof on treating myocardial infarction - Google Patents

Abstract

Provided is a pharmaceutical preparation for myocardial infarction including amniotic fluid-derived cardiomyocytes, which express cardiac troponin T but not MHC class II antigen. Also provided is a use of a pharmaceutical preparation including amniotic fluid-derived cardiomyocytes in the manufacture of a medicament for treating myocardial infarction in a subject in need thereof.

Description

Medicinal preparation of amniotic fluid-derived cardiomyocytes and its use in the treatment of myocardial infarction

The present disclosure relates to a pharmaceutical preparation comprising amniotic fluid-derived cardiomyocytes and its use in treating myocardial infarction.

As terminally differentiated cells, cardiomyocytes are permanent cells that cannot regenerate. After myocardial infarction, although the myocardial precursor cells contained in the myocardium will undergo some compensatory repair through division and proliferation, the number is small and the proliferation ability is low, so the repair takes a long time, and the myocardial tissue cannot be repaired in a timely and effective manner. , and cannot meet the needs of myocardial tissue regeneration in a short period of time, resulting in the final fibrosis of necrotic myocardial tissue, thereby reducing the function of the heart.

The use of exogenous cardiomyocyte transplantation has been confirmed in large animal experiments after myocardial infarction to significantly improve the function of the heart, but allogeneic transplantation is accompanied by severe immune rejection, so that the transplanted animals must be injected with very high doses of anti-rejection drugs, except In addition to the risk of liver toxicity, it also limits the possibility of clinical application.

In addition, in theory, if the patient's own somatic cells are used to reprogram induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) and differentiated into cardiomyocytes, the therapeutic effect will be achieved without rejection. However, studies have pointed out that in the process of reprogramming self-body cells into induced pluripotent stem cells, the genome may be rearranged, so that after transplantation, autologous immune cells cannot recognize and attack the transplant, and eventually produce transplanted cells. immune rejection. In addition, since somatic cells are reprogrammed to differentiate into cardiomyocytes, it usually takes half a year to a year, and it will be too late for acute patients.

In view of this, there is still a need to provide a method for improving heart function to solve the aforementioned problems caused by the current use of allograft transplantation.

The present disclosure provides a pharmaceutical preparation for myocardial infarction and its use in the preparation of a medicament for treating myocardial infarction, wherein the pharmaceutical preparation comprises amniotic fluid-derived cardiomyocytes and pharmaceutically acceptable excipients thereof.

In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes are derived from amniotic fluid stem cells. In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes are differentiated from induced pluripotent stem cells generated from amniotic fluid stem cells.

In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes express cardiac troponin T (cTNT) and do not express MHC class H antigen. In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes produce spontaneous contraction.

In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes are allogeneic relative to the individual system to which the cells are administered. In some specific embodiments of the present disclosure, the The amniotic fluid-derived cardiomyocyte line is derived from this allogeneic amniotic fluid stem cell. In some embodiments of the present disclosure, the amniotic fluid stem cells are isolated from amniotic fluid collected from the allogeneic body at 14 to 16 weeks of gestation or at the end of gestation.

In some embodiments of the present disclosure, the pharmaceutical preparation is formulated into an injectable dosage form. In some embodiments of the present disclosure, the pharmaceutical formulation is used for intramyocardial injection to deliver the amniotic fluid-derived cardiomyocytes to the individual to whom the cells are to be administered.

The present disclosure also provides the use of the above-mentioned amniotic fluid-derived cardiomyocytes for preparing a pharmaceutical preparation for treating myocardial infarction in an individual in need thereof, and the use of the pharmaceutical preparation for preparing a medicament for treating myocardial infarction. In some embodiments of the present disclosure, the drug is used for myocardial regeneration to treat myocardial infarction.

In some embodiments of the present disclosure, the amniotic fluid-derived cardiomyocytes are delivered to the individual by intramyocardial injection. In some embodiments, the injection comprises multiple injections at different sites within the myocardium.

In some specific embodiments of the present disclosure, the amniotic fluid-derived cardiomyocyte line is cryopreserved, and can be directly used for intramyocardial injection after thawing.

In some specific embodiments of the present disclosure, the effective amount of the amniotic fluid-derived cardiomyocytes is 1.0×10 ⁵ to 1.0×10 ⁹ cells, for example, the effective amount is 2.0×10 ⁵ to 1.0×10 ⁹ cells, 5.0×10 ⁵ to 5.0×10 ⁸ cells, 1.0×10 ⁶ to 5.0×10 ⁸ cells, or 1.0×10 ⁶ to 1.0×10 ⁷ cells. In some embodiments, the pharmaceutical formulations of the present disclosure may comprise 1.0×10 ⁵ to 1.0×10 ⁹ amniotic fluid-derived cardiomyocytes, for example, 2.0×10 ⁵ , 5.0×10 ⁵ , 1.0×10 ⁶ , 2.0×10 ⁶ , 5.0×10 ⁶ , 1.0×10 ⁷ , 2.0×10 ⁷ , 5.0×10 ⁷ , 1.0×10 ⁸ , 2.0×10 ⁸ and 5.0×10 ⁸ amniotic fluid-derived cardiomyocytes.

The present disclosure provides a pharmaceutical preparation comprising amniotic fluid-derived cardiomyocytes and its use in treating myocardial infarction. The amniotic fluid-derived cardiomyocytes do not exhibit the second major histophase The characteristics of capacitive antigens can avoid the recognition of immune cells and avoid immune rejection after allogeneic cell transplantation. Furthermore, subjects using the pharmaceutical formulation for myocardial infarction of the present disclosure had reduced cardiac fibrosis and improved cardiac function compared to those who did not use the pharmaceutical formulation of the present disclosure. Furthermore, the amniotic fluid-derived cardiomyocytes disclosed in the present disclosure still maintain a certain activity after repeated freezing-thawing several times, so it is beneficial to prepare medicines or medical materials for effectively treating myocardial infarction.

Figure 1 shows a schematic diagram of the cardiomyocyte differentiation process. hPSCs: human induced pluripotent stem cells; RPMI, B27: culture medium; Chir99021: glycogen synthesis kinase 3 inhibitor Chiron 99021; IWR: Wnt pathway inhibitor IWR-1.

2A to 2C show the results of measuring cell marker characteristics of undifferentiated and differentiated stem cells using flow cytometry. OCT4, Nanog, SSEA4: cell markers of pluripotent stem cells; cTnT: cardiac specific markers; HLA-ABC, HLA-DR: immune-related surface markers; hAFSCs: human amniotic fluid stem cells; hAFSC-iPSCs: induced from human amniotic fluid stem cells Sexual pluripotent stem cells; hAFSC-iPSC-CMs: human amniotic fluid-derived cardiomyocytes; hESCs: human embryonic stem cells; hESC-CMs: cardiomyocytes differentiated from human embryonic stem cells.

Figure 3 shows the contractile performance of cardiomyocytes. hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes; hESC-CM: cardiomyocytes differentiated from human embryonic stem cells. Each value is expressed as mean ± standard error (n = 5).

Figure 4 shows the density and membrane potential of voltage-gated Na ⁺ current (upper row and lower row right graph) and voltage-gated L-type Ca ²⁺ current (upper row left graph and lower row left graph) in cardiomyocytes average relationship. hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes; hESC-CM: cardiomyocytes differentiated from human embryonic stem cells. Each value is expressed as mean ± standard error (n = 8).

5A to 5D show the effect of amniotic fluid-derived cardiomyocyte transplantation on improving cardiac function in myocardial infarction animals. Figures 5A and 5B show that animals with myocardial infarction had reduced cardiac function, and the cardiac function was significantly improved after amniotic fluid-derived cardiomyocyte transplantation. Figure 5C and Figure 5D show the changes in LVEF and LVFS between 2 days after myocardial infarction surgery and 3 weeks after amniotic fluid-derived cardiomyocyte transplantation. LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening; MI: myocardial infarction; hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes.

Figure 6A and Figure 6B show the staining results of heart tissue. In Fig. 6A, the fibrotic infarct area is in red, and the healthy myocardium is in green, and the scale bar is 2 mm. In Fig. 6B, several amniotic fluid-derived cardiomyocyte grafts appeared in the infarct area, and these grafts could be stained by HLA-ABC antibody; the scale bar of conjugated focal immunofluorescence is 50 μm. hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes; HLA-ABC: immune-related surface markers; DAPI: 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole ).

Figures 7A-7C show graft integration, proliferation and apoptosis. Figure 7A shows myocardial regeneration in the infarcted area (dotted line) caused by amniotic fluid-derived cardiomyocytes; the lower row of images is a magnified image of the framed area in the upper row of images; the scale bars of the conjugate immunofluorescence are 50 μm (upper row) and 200 μm (lower row). Fig. 7B shows the hyperplasia of amniotic fluid-derived cardiomyocytes, and the arrows indicate the nuclei of hyperplastic cardiomyocytes; the scale bar is 50 μm. In FIG. 7C , the arrow indicates apoptosis; the scale bar is 50 μm. cTnT: cardiac specific marker; Cx43: connexin 43; DAPI: 4',6-diamidino-2-phenylindole; hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes.

Figure 8 shows the immune response in the infarct area, the scale bar is 50 μm. CD3, CD20, CD68: Cell surface markers, respectively representing CD3 ⁺ T lymphocytes, CD20 ⁺ B lymphocytes, and CD68 ⁺ macrophages. hAFSC-iPSC-CM: human amniotic fluid-derived cardiomyocytes.

The following specific implementations are used to illustrate the technical content of this disclosure. After reading the disclosure of this specification, those skilled in the art can easily understand its advantages and effects. Furthermore, all ranges and values herein are inclusive and combinable. Any numerical value or point, such as any integer, falling within a range stated herein can be used as a minimum or maximum value to derive the underlying range.

As used in the specification and appended claims, the singular forms "a" and "the" include pluralities, and the term "or" includes "and/or" unless otherwise stated herein.

The present disclosure provides a pharmaceutical preparation comprising amniotic fluid-derived cardiomyocytes and its use in treating myocardial infarction. The amniotic fluid-derived cardiomyocyte line is derived from amniotic fluid stem cells that can be safely harvested during routine amniocentesis during pregnancy or during caesarean section. Generally speaking, the fetus and the surrounding placental tissue can be protected from the attack of maternal immune cells through various mechanisms. Since amniotic fluid stem cells themselves exist in the amniotic fluid during pregnancy, amniotic fluid stem cells also have similar characteristics. The special proteins HLA-G and HLA-E that regulate the immune response do not express the second type of major histocompatibility antigen, so to a certain extent, they can avoid the recognition of host T cells and avoid immune rejection.

Cardiomyocytes differentiated from amniotic fluid stem cells (that is, amniotic fluid-derived cardiomyocytes) still retain the immune tolerance of amniotic fluid stem cells. Because they do not express type II major histocompatibility antigen, amniotic fluid-derived cardiomyocytes have certain To a certain extent, it can avoid the identification of host immune cells, and at the same time, it can avoid the occurrence of immune rejection.

Amniotic fluid-derived cardiomyocytes are functional, can produce spontaneous contraction, cell membrane ion currents, etc. during in vitro cell culture, and exhibit cardiac-specific markers (such as cTNT), And because it has the characteristic of avoiding the recognition of host immune cells, it does not need to go through gene pairing, so the cryopreserved cells can be directly thawed and then treated. Therefore, after myocardial infarction, cell therapy can be given immediately to replace necrotic myocardial tissue and promote myocardial regeneration, thereby reducing the risk of subsequent heart failure.

The pharmaceutical preparation of the present disclosure can be used for intramyocardial injection, so as to directly deliver the amniotic fluid-derived cardiomyocytes to the ischemic injury area in the heart, so as to achieve the highest therapeutic effect and avoid the loss or stripping of the graft. In some specific embodiments of the present disclosure, the pharmaceutical preparation is directly used for intramyocardial injection after thawing to treat myocardial infarction.

As used herein, the term "treatment" refers to attempting to achieve a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of total or partial prevention of the disease or its symptoms, or the effect may be in terms of complete or partial cure, alleviation, alleviation, remedy, amelioration of the disease or adverse effects attributable to the disease is therapeutic.

As used herein, the terms "individual" and "patient" are used interchangeably to refer to a human or animal. Examples of individuals include, but are not limited to, humans, monkeys, mice, rats, woodchucks, ferrets, rabbits, hamsters, cows, horses, pigs, deer, dogs, cats, foxes, wolves, chickens, cassowaries, Ostrich, and fish. In some embodiments of the present disclosure, the individual is a mammal, such as a primate, eg, a human.

In some embodiments of the present disclosure, the pharmaceutical preparation further includes pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients can be diluents, disintegrants, binders, lubricants, glidants, surfactants or any combination thereof.

In some embodiments of the present disclosure, the pharmaceutical preparation is for intramyocardial injection. The volume injected can be adjusted as appropriate due to the space of the tissue to be treated without causing discomfort to the patient. In some embodiments, the pharmaceutical formulations of the present disclosure are at less than about 10.0 Injection into a subject in a volume of milliliters (e.g., less than about 5.0 milliliters, less than about 1.0 milliliters, less than about 0.8 milliliters, less than about 0.5 milliliters, or less than about 0.1 milliliters) to deliver an effective amount of amniotic fluid-derived cardiomyocytes .

As used herein, the term "effective amount" refers to the amount of an active agent (e.g., amniotic fluid-derived cardiomyocytes) required to confer a desired therapeutic effect (e.g., a desired improvement in heart function) on a treated subject . As known to those skilled in the art, effective dosages will vary depending on the route of administration, excipient usage, possibility of co-administration with other therapeutic methods, and the condition to be treated.

In some specific embodiments of the present disclosure, the effective amount of the amniotic fluid-derived cardiomyocytes is 1.0×10 ⁵ to 1.0×10 ⁹ cells, for example, the effective amount is 2.0×10 ⁵ to 1.0×10 ⁹ cells, 5.0×10 ⁵ to 5.0×10 ⁸ cells, 1.0×10 ⁶ to 5.0×10 ⁸ cells, or 1.0×10 ⁶ to 1.0×10 ⁷ cells. In some embodiments, the pharmaceutical formulations of the present disclosure may comprise 1.0×10 ⁵ to 1.0×10 ⁹ amniotic fluid-derived cardiomyocytes, for example, 2.0×10 ⁵ , 5.0×10 ⁵ , 1.0×10 ⁶ , 2.0×10 ⁶ , 5.0×10 ⁶ , 1.0×10 ⁷ , 2.0×10 ⁷ , 5.0×10 ⁷ , 1.0×10 ⁸ , 2.0×10 ⁸ and 5.0×10 ⁸ amniotic fluid-derived cardiomyocytes.

In some embodiments of the present disclosure, after the amniotic fluid-derived cardiomyocytes are administered to the individual, they can persist in the individual for at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, About 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks. In some embodiments, the amniotic fluid-derived cardiomyocytes persist in the individual for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months after administration to the individual , about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.

The features and functions of the present disclosure are further described below through specific specific examples, but are not intended to limit the scope of the present disclosure.

Example

Example 1: Isolation of amniotic fluid stem cells

First, amniotic fluid was collected through routine amniocentesis examination in the second trimester (14 weeks to 16 weeks) or at the end of pregnancy, and centrifuged at 300×g for 5 minutes. After removing the supernatant, the cell pellets were washed with phosphate buffered saline (PBS) and dissolved in ammonium chloride to lyse remaining erythrocytes. Next, the cell solution was incubated at 4°C for 20 minutes, and then centrifuged again. The cells after centrifugation were then cultured in α-MEM medium ( alpha minimal essential medium; 11900-024; Gibco Invitrogen, Waltham, MA, USA) in an environment of 37°C and 5% CO ₂ .

After attachment, cells were visible after 4 to 7 days, followed by medium change. When the cells reached 90% confluency, detach the cells using 0.05% trypsin and seed into other culture plates or store frozen.

Example 2: Differentiation from amniotic fluid stem cells into cardiomyocytes

The amniotic fluid stem cells obtained in Example 1 were induced into induced pluripotent stem cells (iPSCs), and were mixed with 15% fetal bovine serum, 1% penicillin/streptomycin and 1% glutamic acid (GlutaMAX Supplement, 35050061 ; Gibco) in α-MEM medium at 37 ° C, 5% CO ₂ environment.

Next, cardiomyocyte differentiation was performed referring to the flow chart shown in FIG. 1 . First, the cells were expanded in α-MEM medium containing 4 ng/mL basic fibroblast growth factor (bFGF; 233-FB; R&D Systems, Minneapolis, MN, USA), and then separated and grown in 1.4×10 The amount of ⁶ cells were seeded in a 6-well culture dish coated with Matrigel, and the day before the monolayer of cells became compact was marked as day -1. The next day (i.e. day 0), remove the medium, and replace with B-27 ( A18956-01 ; A18956-01; Gibco Invitrogen) RPMI medium (11875119; Gibco Invitrogen) was replaced, cultured at 37°C, 5% CO ₂ for 2 days, and then replaced with new RPMI medium for another 24 hours.

On the 3rd day, the medium was replaced with RPMI medium containing 5 μM of Wnt pathway inhibitor IWR-1 (I0161; Sigma) and B-27 without insulin, and after 2 days of culture, the medium was replaced with B-27 (insulin-free). ) in RPMI medium; on day 7, the medium was replaced with RPMI medium containing B-27 (with insulin) (17504-044; Gibco Invitrogen), and then the medium was changed every 2 days. On day 12, the medium was changed to RPMI medium (without glucose) containing B-27 (with insulin) to purify cardiomyocytes, and the medium was changed every 2 days thereafter. On day 16, the medium was changed to RPMI medium containing B-27 (with insulin).

Example 3: Characteristics of amniotic fluid-derived cardiomyocytes

The cell marker characteristics of cardiomyocytes differentiated from day 0 to day 21 in Example 2 were measured by flow cytometry, wherein cardiomyocytes differentiated from human embryonic stem cells (RUES2 cell line) were used as a control group.

First, cells were isolated from the culture medium, and then stained with various fluorescently-labeled antibodies such as OCT4, Nanog, SSEA4, HLA-ABC, HLA-DR, and cTnT (BD Biosciences, Franklin Lakes, NJ, USA). Cells were permeabilized with permeabilization buffer (554723; BD Biosciences) prior to intracellular cTnT staining. The fluorescence characteristics of the cells were then analyzed using a flow cytometer BD FACS Canto II (BD Biosciences).

The results are shown in Figure 2A to Figure 2C, undifferentiated amniotic fluid stem cells (hAFSC), iPSCs derived from amniotic fluid stem cells (hAFSC-iPSC) and human embryonic stem cells (hESC) all express cell markers of pluripotent stem cells, namely OCT4, Nanog and SSEA4, showing that the three stem cells All have pluripotency; in addition, these three stem cells do not express the heart-specific molecule cTnT (ie, cardiac troponin T). Stem cells differentiated into cardiomyocytes showed only low levels of pluripotent stem cell markers but high expression of cTnT compared to undifferentiated stem cells.

Furthermore, Figure 2A to Figure 2C show that hAFSC and hAFSC-iPSC express the first major histocompatibility antigen (MHC class I antigen) (that is, human leukocyte antigens HLA-A, B, C), but do not express Type II major histocompatibility antigen (eg, human leukocyte antigen HLA-DR). However, hESCs express both type I and type II major histocompatibility antigens. It is also worth noting that the differentiated amniotic fluid-derived cardiomyocytes still do not express HLA-DR, indicating that the cells have the potential to avoid immune cell recognition or attack.

Next, in order to test the contractility of cardiomyocytes, an inverted microscope equipped with a camera and capable of slow-motion shooting (120 frames per second) was used to record cardiomyocytes differentiated at 0, 15, 20, 30 and 50 days beating. The filmed films were analyzed using the automated non-profit software MUSCLEMOTION and the contraction force profile (ie contraction magnitude and velocity) was measured. The results are shown in Figure 3. The amniotic fluid-derived cardiomyocytes exhibited contractile properties similar to those of cardiomyocytes differentiated from human embryonic stem cells (hESC-CM), such as contraction amplitude, contraction time, contraction velocity, and relaxation velocity.

Example 4: Measurement of electrophysiological properties of amniotic fluid-derived cardiomyocytes

Cardiomyocytes have connexin 43 gap junction channels and can contract spontaneously. In order to confirm that the amniotic fluid-derived cardiomyocytes indeed have the function of cardiomyocytes, an aliquot of the cell suspension (separated with 1% trypsin/ethylenediaminetetraacetic acid (EDTA) solution) was placed on an inverted fluorescent microscope ( CKX-41; Olympus, Yuanyu, Taipei, Taiwan) on the recording chamber of the stage. To avoid mechanical noise, a microscope equipped with a digital video recording system (DCR TRV30; Sony, Tokyo, Japan) equipped with 1,500× and 40× objective lenses was placed on an anti-vibration air table, and an RK-400 (Bio-Logic, Claix, France) or Axopatch 200B (Molecular Devices, Sunnyvale, CA, USA) amplifier for patch-clamp recording.

To measure the voltage-gated Na ⁺ current ( I _Na ) and the voltage-gated L-type Ca ²⁺ current ( I _Ca,L ), cells were immersed in Ca ²⁺ -free Tyrode's solution for membrane Piece-clamp experiments were performed, and current and potential signals were monitored using a digital oscilloscope (model 1602; PChome eBay Co., Ltd., Taipei, Taiwan). To elicit INa , test cells were _depolarized from -80 mV to various potentials in the range of -100 mV and +40 mV in increments of 10 mV. To record I _Ca,L , cells were voltage clamped at -50 mV and a depolarizing command potential between -50 mV and +40 mV was applied.

Data were collected through an acquisition port (Digidata-1440A; Molecular Devices, Sunnyvale, CA) and subsequently analyzed using pCLAMP 10.7 (Molecular Devices) or 64-bit OriginPro 2016 (Microcal; Scientific Formosa, Inc., Kaohsiung, Taiwan). Through the digital-to-analog converter of Digidata-1440A device, pCLAMP generates the average relationship between I _Na and I _{Ca, L} density and membrane potential.

The results are shown in Figure 4. On the 7th day (week 1) of cardiomyocyte differentiation, when the cardiomyocytes did not contract spontaneously, the currents of I _Na and I _Ca,L between amniotic fluid-derived cardiomyocytes and hESC-CM On the 21st day (3rd week), cardiomyocytes contracted spontaneously, and had higher I _Na and I _{Ca, L} currents, and the membrane potential of these cardiomyocytes was about -60mV to -65mV.

Example 5: Effect of amniotic fluid-derived cardiomyocytes on myocardial infarction

In this embodiment, myocardial infarction (MI) rats were used to test the efficacy of amniotic fluid-derived cardiomyocytes on myocardial infarction. All animal experiments were approved by the Animal Care and Use Committee of National Cheng Kung University and performed in accordance with the Guide for the Care and Use of Animals.

First, about 12-week-old SD rats (about 350g to 400g) were anesthetized by intraperitoneal injection of 1mg/kg Zoletil 50 (Virbac, France), intubated and ventilated by a ventilator (model 683; Harvard, USA). ) was mechanically ventilated at a frequency of 80 times/min, and a volume of 200 μL was maintained, thereby maintaining adequate respiration of the rat.

This is followed by a thoracotomy to expose the heart and an ischemia-reperfusion (I-R) procedure, that is, by complete occlusion of the mid-left anterior descending coronary artery (LAD) flow60 minutes to induce myocardial infarction. Then use 6-0 silk suture. After reperfusion of the LAD blood flow, the thoracotomy wound was closed with 4-0 polypropylene suture. The animals were placed in a warm environment to maintain their core temperature at approximately 35°C to 37°C until recovery from anesthesia, and the animals were closely monitored.

The amniotic fluid-derived cardiomyocytes used for intramyocardial transplantation refer to the 21st day of differentiation cells obtained in Example 2. These amniotic fluid-derived cardiomyocytes were suspended in a pro-survival cocktail medium containing the following components: Matrigel with reduced growth factors (50%; BD; 354230); 100 μM ZVAD (62761; Calbiochem ; Massachusetts, USA); 50 μM Bcl-XL BH4 (197217; Calbiochem; Massachusetts, USA); 200 nM cyclosporine A (PHR1092; Sigma, Massachusetts, USA); 100 ng/mL insulin-like growth factor 1 (590904; Biolegemd; California, USA); 50 μM pinacidil (P154; Sigma). Keep cells on ice until injection.

On the 4th day after IR surgery, another thoracotomy was performed (this day was marked as day 0), and intramyocardial transplantation of amniotic fluid-derived cardiomyocytes (1.0× ¹⁰⁷ cells) was performed using a 0.3 mL insulin syringe and a 31-gauge needle /kg, dissolved in 80μL pro-survival cocktail medium, n=6). The transplantation was performed in 2 injections, ie, each injection was 40 μL of a solution of approximately 5.0×10 ⁶ cells. The control group was injected with vehicle only (ie, culture medium without cells) (40 μL, 2 injections). Two intramyocardial injections were performed according to the following procedure: one injection in the center of the infarcted area, and one injection in the border region of the infarcted area. Then, the open chest surgery wound is closed using 4-0 polypropylene sutures, after which pain medication and antibiotics are administered to reduce post-operative pain and the risk of infection.

In general, immunosuppressive therapy is combined with methylprednisolone (methylprednisolone; 2 mg/kg/day, intraperitoneal injection) and cyclosporin A (cyclosporin A, CsA; 15 mg/kg/day, subcutaneous injection). However, in this example, starting from the day before cell transplantation (Day -1), all rats received only a low dose of CsA (10 mg/kg/day, subcutaneous injection) until sacrifice (intramyocardial injection 3 weeks later).

In addition, another 4 rats underwent thoracotomy, but did not undergo I-R surgery and intramyocardial transplantation, and this group of rats was used as a sham operation group. The rats in this group were anesthetized with 1 mg/kg of Zoletil 50 (Virbac, France), intubated and mechanically ventilated by a ventilator (model 683; Harvard, USA) at a frequency of 70 times/min, and a volume of 200 μL was maintained. , thereby maintaining adequate respiration of the rat.

Cardiac function in rats was assessed by repeat echocardiography 1 day before, 2 days after and 4 weeks after I-R surgery (ie, 3 weeks after intramyocardial injection). Specifically, rats were lightly anesthetized with isoflurane inhalation (Novaplus), and scanned by transthoracic echocardiography (vevo 770; VisualSonics, Toronto, Canada) with a 10 MHz probe. The obtained left ventricular end-diastolic dimension (LVEDD), end-diastolic left ventricular volume (left ventricular end-diastolic volume, LVEDV), Left ventricular end-systolic dimension (LVESD) and left ventricular end-systolic volume (LVESV) were used to measure left ventricular ejection fraction (LVEF) and left ventricular Short axis shortening rate (left ventricular fractional shortening, LVFS), its calculation formula is as follows:

LVEF=[(LVEDV-LVESV)/LVEDV]×100(%);

LVFS=[(LVEDD-LVESD)/LVEDD]×100(%).

Also, on day 21, rats were sacrificed by intravenous injection of phenobarbital and phenytoin sodium (Beuthanasia-D) and hearts were harvested. The heart was washed with sterile normal saline, and then perfused with 0.9% normal saline and 4% paraformaldehyde, and then the left and right atrium and right ventricle were taken out. The left ventricle was sectioned parallel to the short axis to a thickness of 1 mm and stained with Picrosirius red/fast green to visualize the infarct/fibrosis area. Infarct size was calculated as Σ(infarct size/block size) and expressed as a percentage of the left ventricle.

Fluorescent staining was performed using primary antibodies against α-actin (ab68194; Abcam), primary antibodies against human-specific HLA-ABC (ab70328; Abcam) and their corresponding secondary fluorescent antibodies (Abcam) to distinguish sheep Water-derived cardiomyocyte grafts. Graft area was calculated as Σ(graft area/infarct area) and expressed as a percentage of infarct area. Fluorescent staining images were acquired with an Olympus BX51 microscope (OLYMPUS, Tokyo, Japan) and quantified using Image J software.

The measurement results of cardiac function are shown in Table 1 below:

The above data can also be seen in Figure 5A and Figure 5B. From these results, it can be seen that there was no significant difference in the left ventricular systolic function of animals in each group before IR surgery. However, 2 days after IR surgery, the LVEF of the control group decreased from 67.2±1.4 to 48.6±1.9 ( p =0.004), and the LVFS decreased from 33.9±3.3 to 15.8±3.4 ( p =0.002); the LVEF of the cell transplantation group decreased from 74.7 ±2.0 decreased to 56.2±1.1 ( p =0.002), and LVFS decreased from 35.8±3.4 to 24.1±1.4 ( p =0.002), showing that MI did cause a decrease in left ventricular systolic function.

After 3 weeks of intramyocardial transplantation, the cell transplantation group showed significantly improved left ventricular systolic function (LVEF from 56.2±1.1 to 65.6±1.1 ( p =0.002), LVFS from 24.1±1.4 to 34.4 ±1.4 ( p =0.006)). Furthermore, referring to FIG. 5C and FIG. 5D , the changes of LVEF and LVFS between 2 days after MI and 4 weeks after MI showed that intramyocardial transplantation of amniotic fluid-derived cardiomyocytes significantly improved the systolic function of the left ventricle.

The results of histological staining in Figure 6A and Figure 6B showed that all MI animals had transmural scars, and the animals that received amniotic fluid-derived cardiomyocyte transplantation showed partial myocardial regeneration and considerable area of the graft. Furthermore, teratoma formation was not observed in the cell transplantation group.

In addition, Figure 7A to Figure 7C show that left ventricle sections were detected by anti-cTnT antibody (ab8295; Abcam), anti-connexin 43 antibody (Cx43; C6219; Sigma), anti-Ki67 antibody (11882s; Cell Signaling Technology) and anti-annexin V (Annexin V) antibody (640945; BD Biosciences) fluorescent staining results. As shown in Figure 7A, connexin 43 was present in the amniotic fluid-derived cardiomyocyte grafts and in the intercalated disc between the graft and the host tissue, showing that the amniotic fluid-derived cardiomyocyte graft tissue was closely related to the graft and Electrical coupling may be formed between the myocardium of the host.

The proliferation and apoptosis of cardiomyocytes were assessed by the expression of Ki67 and annexin V, respectively. As shown in Figure 7B, compared with the control group, there were more cell cycle activities in the cell transplantation group, implying cell proliferation; however, as shown in Figure 7C, there was no difference in cell apoptosis between the control group and the cell transplantation group. No significant difference.

In order to further confirm the immune rejection after transplantation, primary antibodies against CD3, CD20 and CD68 (Abcam), EnVision detection system peroxidase/diaminobenzidine (DAB) kit (DAKO, K5007) were used And the secondary antibody of horseradish peroxidase (HRP) was used for fluorescent staining of left ventricle slices.

The results are shown in Figure 8. Similar to the sham operation group and the control group, only a small amount of CD3 ⁺ T lymphocytes, CD20 ⁺ B lymphocytes, and CD68 ⁺ macrophages appeared in the cell transplantation group, indicating that the amniotic fluid-derived cardiomyocyte transplantation Does not cause serious immune rejection.

The above-mentioned embodiments are used to illustrate the principles and functions of the present disclosure, but not to limit the present disclosure. Anyone skilled in the art can modify the above embodiments without departing from the scope of this disclosure. Therefore, the scope of protection of the rights of this disclosure should be listed in the scope of the patent application described later.

Claims (9)

A pharmaceutical preparation for myocardial infarction, comprising amniotic fluid-derived cardiomyocytes and pharmaceutically acceptable excipients thereof, wherein the amniotic fluid-derived cardiomyocytes do not express the second major histocompatibility antigen. The pharmaceutical preparation according to claim 1, wherein the amniotic fluid-derived cardiomyocytes express cardiac rotin T. The pharmaceutical preparation according to claim 1, wherein the amniotic fluid-derived cardiomyocytes produce spontaneous contraction. Use of a pharmaceutical preparation comprising amniotic fluid-derived cardiomyocytes for the preparation of a medicament for treating myocardial infarction in an individual in need thereof, wherein the amniotic fluid-derived cardiomyocytes do not express type II major histocompatibility antigen, the amniotic fluid-derived The water-derived cardiomyocytes are induced pluripotent stem cells differentiated from amniotic fluid stem cells, and the amniotic fluid stem cells are isolated from allogeneic amniotic fluid of the individual. The use as described in Claim 4, wherein the amniotic fluid stem cells are isolated from the amniotic fluid collected from the allogeneic body at 14 to 16 weeks of pregnancy or at the end of pregnancy. The use according to claim 4, wherein the amniotic fluid-derived cardiomyocytes are delivered to the individual through intramyocardial injection. The use as described in claim 6, wherein the amniotic fluid-derived cardiomyocytes are directly injected into the myocardium after thawing. The use as described in Claim 4, wherein the effective amount of the amniotic fluid-derived cardiomyocytes is 1.0×10 ⁵ to 1.0×10 ⁹ cells. The use as described in Claim 4, wherein the drug is used for myocardial regeneration to treat the myocardial infarction.

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