Active ingredients that promote stem cell differentiation and serve as post-myocardial infarction care.

Abstract

This case discloses the method of using Codonopsis pilosula extract to promote stem cell differentiation, the use of Codonopsis pilosula extract as a stem cell differentiation promoter, and the use of Codonopsis pilosula extract for post-healing care of myocardial infarction.

Description

Technical Field

[0001] This case concerns novel uses of Codonopsis pilosula extract, particularly its use as a stem cell differentiation promoter and for post-infarction care. Background Technology

[0002] Heart failure is a condition caused by abnormalities in the structure or function of the heart, posing a potential threat to life. There are many causes of heart failure, most of which stem from myocardial infarction and myocardial ischemia, leading to damage or death of myocardial tissue. In industrialized countries, the risk of heart disease increases with age and other risk factors, such as diabetes and obesity.

[0003] Currently, the main treatments for heart failure include medication and surgical intervention, but both have limitations. These treatments cannot restore damaged myocardial tissue and are more likely to cause other side effects (Li S.C., Wang L., Jiang H., Acevedo J., Chang AC, and Loudon WG (2009) Stemcell engineering for treatment of heart diseases: Potentials and challenges. Cell Biol. Int. 33, 255-67.). Therefore, alternative therapies to restore damaged myocardial tissue have emerged.

[0004] Stem cells, with their differentiation and regeneration capabilities, are considered a potential treatment for heart failure (Ruvinov E., Dvir T., Leor J. and Cohen S. (2008) Myocardial repair: from salvage to tissue reconstruction. Expert Rev. Cardiovasc. Ther. 6, 669-686; Segers VF and Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451, 937-942; and Zhang J., Wilson GF, Soerens AG, Koonce CH, Yu J., Palecek SP, Thomson JA and Kamp TJ (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circulation Research 104, e30-e41.). Stem cells can be induced into primary cardiac progenitor cells in vitro and differentiate into cardiomyocytes, making them a potential candidate for replacing infarcted or damaged heart tissue and restoring its function. However, transplantation of non-primary cardiac cells may lead to tumor formation, causing potentially fatal problems. Other issues that need to be addressed include stem cell population expansion, homing and loss of introduced stem cells, functional integration of stem cells, and the appropriate routes for stem cell infusion.

[0005] Therefore, how to effectively promote the differentiation of stem cells into cells with myocardial function and reduce the occurrence of side effects has become an important issue in this field. Summary of the Invention

[0006] This invention was developed to address the aforementioned issues. It utilizes natural herbal extracts to promote stem cell differentiation, forming cells with cardiomyogenic activity to replace damaged or dead cardiomyocytes caused by myocardial infarction or ischemia. Furthermore, through animal models, this invention demonstrates that natural herbal extracts can improve the function of cardiomyocytes after infarction.

[0007] More specifically, this disclosure reveals the following invention:

[0008] (1) A method for promoting stem cell differentiation, comprising: adding Codonopsis pilosula extract as a differentiation promoter to the culture medium in which the stem cells are cultured.

[0009] (2) The use of a Codonopsis pilosula extract as a stem cell differentiation promoter.

[0010] (3) The use of a Codonopsis pilosula extract for post-healing care of myocardial infarction. Attached Figure Description

[0011] Figure 1A Bright-field imaging (left) of contractile cardiomyocytes that have formed an embryonic body (EB) and the expression of green fluorescent protein (eGFP) (middle). The superimposed images (right) show the contractile cell population expressing eGFP.

[0012] Figure 1B The images show contractile cardiomyocytes stained with immunocytochemistry, confirming that the contractile cells expressing eGFP are cardiomyocytes.

[0013] Figure 2 Bright-field imaging showing EMG8 transgenic stem cells spontaneously differentiating into cardiomyocytes without forming EB (left image) and eGFP expression (middle image), and the superimposed images (right image) confirm that the differentiated cardiomyocytes express eGFP.

[0014] Figure 3A The study showed the effect of Codonopsis pilosula extract on the differentiation of EMG8 transgenic stem cells, demonstrating that Codonopsis pilosula extract can promote the differentiation of EMG8 transgenic stem cells into cardiomyocytes.

[0015] Figure 3B This study demonstrates the dose-related effect of Codonopsis pilosula extract on the differentiation of EMG8 transgenic stem cells.

[0016] Figure 4A This study demonstrates the effect of Codonopsis pilosula extract on the fractional shortening (FS) of cardiac function in a rat model of myocardial infarction.

[0017] Figure 4B This study demonstrates the effect of Codonopsis pilosula extract on the fractional area contraction (FAC) of cardiac function in a rat model of myocardial infarction.

[0018] Figure 4C This study demonstrates the effect of Codonopsis pilosula extract on cardiac function, specifically ventricular ejection fraction (EF), in a rat model of myocardial infarction.

[0019] Figure 5 This illustrates the pMGN22 carrier structure constructed in one embodiment of the present invention. Detailed Implementation

[0020] This disclosure uses Codonopsis pilosula (France) Nannf. extract as a stem cell differentiation promoter and as an active ingredient for post-healing care of myocardial infarction.

[0021] Codonopsis pilosula (France) Nannf. is the dried root of a perennial herb belonging to the Campanulaceae family. In traditional Chinese medicine, it is considered to have the effects of tonifying the middle energizer and replenishing qi, strengthening the spleen and stomach, and is one of the commonly used Chinese medicinal herbs (Lin Zichao et al., A Materia Medica Investigation of Codonopsis pilosula, J. Chin. Med 18(1,2):51-64, 2007). Codonopsis pilosula is often used in combination with other Chinese herbal medicines to achieve effects such as tonifying qi and strengthening the stomach. However, there is no literature on the effects of Codonopsis pilosula in promoting stem cell differentiation or in promoting recovery after myocardial infarction. Furthermore, there is no record of the possibility of Codonopsis pilosula causing toxicity in animals. Based on the view that it has been used for a long time without animal toxicity, the use of Codonopsis pilosula can reduce or eliminate the side effects or toxicity that may be caused by the use of chemically synthesized drugs.

[0022] This disclosure uses Codonopsis pilosula extract as a stem cell differentiation promoter and as an active ingredient for post-infarction care. More specifically, the Codonopsis pilosula extract used in this disclosure is derived from dried Codonopsis pilosula powder extracted with water. The ratio of dried Codonopsis pilosula powder to water used for extraction is not particularly limited, and can be 1:5 to 1:20 (Codonopsis pilosula:water by weight). In one embodiment, the weight ratio of Codonopsis pilosula to water is 1:10. The extraction temperature is usually above room temperature, such as 80-100°C. In one embodiment, extraction is performed at 90-100°C. The extraction method can be hot backflow extraction, performed for 2-5 hours.

[0023] To ensure long-term and stable preservation, the Codonopsis pilosula extract disclosed herein can be further concentrated and freeze-dried for storage in dry powder form. Before use, it can be reconstituted in water. The reconstituted concentration can be 10~1000 μg / ml; in one embodiment disclosed herein, the reconstituted concentration is 100 μg / ml.

[0024] The stem cells described in this disclosure can be derived from embryonic stem cells of non-human mammals or adult stem cells of mammals, such as umbilical cord blood stem cells, bone marrow stem cells, and adult peripheral blood stem cells. Since embryonic stem cells from non-human mammals are obtained from the hollow, tiny blastocysts of embryos developing at 4-5 days of age, they possess the potential to differentiate into all the cells and tissues required to develop into a complete individual, making them a preferred differentiation target. Furthermore, the mammals that can be used in this application include, for example, mice, rabbits, pigs, cattle, dogs, cats, monkeys, apes, or humans, without particular limitation.

[0025] To confirm the stem cell differentiation-promoting effect of Codonopsis pilosula extract, this disclosure specifically designed a transgenic stem cell line with the promoter of the mouse α-cardiac myosin heavy chain (α-MHC) gene and the green fluorescent protein (eGFP) gene. The α-MHC gene is a cardiomyocyte-specific gene expressed in the early stages of heart development (Morkin E. (2000) Control of cardiac myosin heavy chain gene expression. Microsc. Res. Tech. 50, 522-531.). Using the transgenic stem cell line designed in this disclosure, the fluorescence expression of the marker protein eGFP can be used to confirm whether the stem cells differentiate into cells with cardiomyogenic activity, thereby detecting the effect of Codonopsis pilosula extract on promoting stem cell differentiation. According to one embodiment of this disclosure, regardless of whether the aforementioned transgenic stem cells form embryoid bodies (EBs), the Codonopsis pilosula extract disclosed herein exhibits an effect of promoting the differentiation of these stem cells into cells with cardiomyogenic activity. On the other hand, the use of other solvent extracts of Codonopsis pilosula, such as ethanol, did not produce the effect of promoting stem cell differentiation (this experiment and data are not shown in this disclosure).

[0026] Furthermore, this disclosure also provides the efficacy of Codonopsis pilosula extract in post-myocardial infarction care. According to one embodiment of this disclosure, Codonopsis pilosula extract has the effect of improving myocardial function in an animal model of myocardial infarction. This effect is reflected in indicators such as fractional shortening (FS), fractional area contraction (FAC), and ventricular ejection fraction (EF).

[0027] According to this disclosure, Codonopsis pilosula extract exhibits cardiogenic activity, which can be used to promote the differentiation of stem cells, especially embryonic stem cells from non-human mammals, into cells with cardiomyogenic activity, thereby producing cardiomyocytes for application in cell therapy. Furthermore, according to this disclosure, Codonopsis pilosula extract has the effect of improving myocardial function in hearts damaged after acute myocardial infarction, and can be effectively used as a maintenance active ingredient for post-myocardial infarction recovery.

[0028] The following describes preferred embodiments of the present invention. For clarity, the following descriptions and drawings are appropriately omitted and simplified. Furthermore, the present invention is not limited to the embodiments described above. Within the scope of the present invention, practitioners can easily modify, add to, or alter the elements of the above embodiments.

[0029] [Example 1]

[0030] Construction of carrier

[0031] The mouse α-MHC promoter (obtained from Jeffrey Robbins (Gulick J., Subramaniam A., Neumann J. and Robbins J. (1991) Isolation and characterization of the mousecardiac myosin heavy chain genes. J. Biol. Chem. 266, 9180-9185.)) was cleaved by BamHI-SalI into a 5.5kb fragment and inserted into the BgIII-SaII restriction enzyme cleavage site (MCS) of pEGFP-1 (Clontech) to construct the vector pMGN22 (as shown in Figure 5).

[0032] Preparation of transgenic stem cell lines

[0033] According to the instructions in the manual, the vector pMGN22 was transfected into mouse stem cell line ES-D3 (ATCC) using lipofectamine 2000 (Invitrogen) to prepare the transgenic stem cell line EMG8.

[0034] Specifically, the surface was coated with 0.1% gelatin (Millipore), and the following were used: stem cell culture medium without feeder cells (containing Dulbecco modified essential medium (DMEM) (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.15 mM α-thioglycerol (monothioglycerol) (ICN Biomedicals Inc.), 15% EScell-qualified fetal bovine serum (Gibco), penicillin G (100 U / ml), streptomycin (100 μg / ml), amphotericin B (250 ng / ml), and 10 3 The prepared transgenic stem cell line EMG8 was screened in 500 μg / ml G418 (Sigma) at ESGRO (Chemicon) with a concentration of 1 U / ml. The resulting EMG8 transgenic stem cell line was then cultured in stem cell medium containing 250 μg / ml G418 at 37°C with 5% CO2. The medium was changed daily.

[0035] α-MHC is a cardiomyocyte-specific gene expressed early in heart development (Morkin E. (2000) Control of cardiac myosin heavy chain gene expression. Microsc. Res. Tech. 50, 522-531.). The transgenic stem cell EMG8 constructed as described above can demonstrate the process of stem cell differentiation into cardiomyocytes.

[0036] Differentiation of mouse stem cell lines

[0037] The obtained EMG8 transgenic stem cell lines were seeded at 2000 cells per well in 96-well Corning plates containing stem cell differentiation medium (containing high-glucose DMEM medium (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM beta-mercaptoethanol (Sigma), 20% fetal bovine serum (Gibco), 100 U / ml penicillin G, 100 μg / ml streptomycin, 250 ng / ml amphotericin B, and 250 μg / ml G418) and coated with 0.1% gelatin (Millipore). The medium was changed daily. After 10 days, the intensity of enhanced green fluorescent protein (eGFP) at an excitation wavelength of 480 nm and an emission wavelength of 519 nm was measured using a SpectraMax M2 microdroplet reader (Molecular Devices).

[0038] On the other hand, the EMG8 transgenic stem cell lines obtained above spontaneously differentiated to form embryonic bodies (EBs) (Hescheler J., Fleischmann BK, Lentini S., Maltsev V.A., Rohwedel J., Wobus AM and Addicks K. (1997) Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. 36, 149-162; and Pucéat M. (2008) Protocols for cardiac differentiation of embryonic stem cells. Methods 45, 168-171.). In short, after 2 days of culture at 5% CO2 and 37°C, hanging droplets of embryonic bodies (EBs) formed on the top of a culture dish (SPL) containing culture medium, with 500 cells in 25 μl of stem cell differentiation medium. These cells were then suspended in stem cell differentiation medium for another 5 days. These embryonic bodies (EBs) were then transferred to six-well discs (Nunces) containing stem cell differentiation medium coated with gelatin for continued differentiation. The contractile and beating behaviors of the EBs were observed using an optical microscope. Images of the contractile behavior of EBs expressing green fluorescent protein (eGFP) were recorded using a Leica DM IRBE microscope equipped with an Andor Luca-R EMCCD.

[0039] Preparation of Codonopsis pilosula extract

[0040] 100g of Codonopsis pilosula (France) Nannf. powder was extracted with hot reflux at 95°C and 1000ml of water for 2.5 hours. The crude extract was then cooled, and a clear extract was separated from it using a suction filter or chromatography. The extract was then concentrated and freeze-dried to form a dry powder for long-term storage. In this example, the extract was redissolved in deionized water to a concentration of 100μg / ml to confirm the cardiogenic activity of the Codonopsis pilosula extract.

[0041] Immunochemical staining

[0042] Embryomorphs (EBs) derived from EMG8, which were seeded onto gelatin-coated slides and cultured for 15 days, were fixed with 4% formaldehyde at room temperature for 20 minutes. They were then permeabilized with 0.5% Triton X-100 for 5 minutes, followed by blocking with 5% healthy goat serum (NGS) / PBS at room temperature for 20 minutes. The EBs were then co-cultured overnight at 4°C with primary antibodies (rabbit anti-Nkx2-5:1:150 [GeneTex] or mouse anti-α-actin IgG 1:1:200 [Enzo]) in 1% NGS / PBS. After washing twice with PBS, the EBs were co-cultured at room temperature with secondary antibodies (goat anti-rabbit Qdot655-conjugated IgG:1:200 [Invitrogen] or goat anti-mouse TRICT-conjugated IgG:1:200 [Jackson ImmunoResearch]) for 1 hour. The stained images were then observed using a Leica DM IRBE microscope equipped with an Andor Luca-R EMCCD.

[0043] laboratory animals

[0044] Three-month-old male Wistar rats (obtained from the Laboratory Animal Center of the College of Medicine, National Cheng Kung University) were used as experimental animals. All animal handling procedures were conducted in accordance with the approvals of the Laboratory Animal Care and Use Committee of National Cheng Kung University and the Institute of Laboratory Animal Resource.

[0045] Preparation of a rat model of myocardial infarction

[0046] After inhaling isoflurane in the anesthesia room, the experimental rats were inserted with a 16G blood catheter and placed on a Harvard ventilator with positive pressure ventilation. The anesthetic was maintained at 2% inhaled isoflurane, and an electrocardiogram (EKG) was performed for monitoring. The left anterior chest and pericardium were incised, and the heart was visualized through the fourth intercostal space. The left anterior descending (LAD) artery was bound with 7-0 prolene suture (Kan CD, Lee HL, and Yang Y.J. (2010) Cell transplantation for myocardial injury: a preliminary comparative study. Cytotherapy 12, 692-700.). During the procedure, myocardial ischemia was confirmed by visual inspection and ST segment assessment using EKG. The rib and subcutaneous wounds were then sutured with 3-0 Vicryl (Ethicon Co, Inc.) and 3-0 Ethilon (Ethicon Co, Inc.). All experimental rats received the same amount of intramuscular antibiotics and anesthetics after surgery.

[0047] Myocardial function assessment

[0048] One week after left anterior descending artery (LAD) ligation, myocardial function and affected myocardial wall volume were measured using echocardiography as baseline data. Rats were anesthetized and placed in the left lateral decubitus position. Short-axis 2D images of the left ventricle (LV) at the mid-papillary level were stored as digital loops, and the endocardial borders were traced to identify the end-systolic (ESA) and end-diastolic (EDA) cavity areas.

[0049] The fractional shortening (FS) is calculated by subtracting the left ventricular end-diastolic dimension (LVESD) from the left ventricular end-systolic dimension (LVEDD) and then dividing by the left ventricular end-systolic dimension (LVEDD) on the M-mode image.

[0050] The fractional area contraction (FAC) is calculated as the percentage of the difference between the end-diastolic volume and the end-systolic volume relative to the end-diastolic volume ([EDA–ESA] / EDA × 100).

[0051] The ventricular ejection fraction (EF) is calculated as the percentage of stroke volume to end-diastolic volume [(SV / EDV) × 100]. Stroke volume (SV) is equivalent to end-diastolic volume (EDV) minus end-systolic volume (ESV).

[0052] Each measurement was performed three times, and the average value was taken. After cardiac ultrasound assessment and LAD restraint, rats were randomly divided into two experimental groups (i.e., a control group and a group given Codonopsis pilosula extract). Myocardial function was assessed by the same cardiac ultrasound by observers who were not informed of the above experimental treatment.

[0053] result

[0054] eGFP expression in contractile cardiomyocytes that have formed embryo-like bodies (EBs)

[0055] On day 15 post-culture (2 days of hanging drop culture, 5 days of suspension culture, and 8 days of fixed culture), eGFP expression during embryoid body (EB) growth was observed using fluorescence microscopy. Figure 1A , 1B As shown. Figure 1A The left image is a bright-field imaging, showing the contractile cell population located within a circular region. Figure 1A The middle image shows the green fluorescence expression of eGFP, which completely overlaps with the bright-field imaging in the left image. Figure 1A The right image shows that these contractile cell populations express eGFP.

[0056] To further confirm the contractile cells expressing eGFP, EB growth was presented using immunocytochemistry. Results are as follows: Figure 1B As shown, contractile cells expressing eGFP were stained with anti-α-actin and anti-Nkx2-5 antibodies. Since α-actin and Nkx2-5 are proteins specifically present in cardiomyocytes, the contractile cells expressing eGFP were confirmed to be cardiomyocytes.

[0057] Cardiomyocytes expressing eGFP were observed in spontaneously differentiated EMG8 cells.

[0058] EMG8 cells, without forming EB, were directly seeded onto stem cell differentiation medium and spontaneously differentiated. Nine days later, the expression of eGFP in the differentiated stem cells was observed using an epifluorescence microscope. The results are as follows: Figure 2 As shown, eGFP-positive cells were present, indicating that EMG8 cells spontaneously differentiated into cardiomyocytes. Based on these results, the cardiogenic activity of Codonopsis pilosula extract can be assessed by seeding EMG8 cells into stem cell differentiation medium containing Codonopsis pilosula extract.

[0059] Codonopsis pilosula extract promotes cardiogenic differentiation of EMG8 cells in a dose-related manner.

[0060] To observe the cardiogenic activity of Codonopsis pilosula extract, EMG8 cells were seeded at 2000 cells per well in a gelatin-coated 96-well dish containing stem cell differentiation medium (Day 1). On Day 2, 0.5 mg / ml of Codonopsis pilosula extract was added to each well, with 5 samples per test. The medium was changed daily. On Day 11, the medium was removed, and the 96-well dish was transferred to a fluorescence microdroplet reader with an excitation wavelength of 480 nm and an emission wavelength of 519 nm for detection. The results are as follows: Figure 3A As shown, the cardiogenic activity of Codonopsis pilosula extract was 24.2%, higher than that of the control group. Furthermore, the cardiogenic activity of Codonopsis pilosula extract on EMG8 cells showed a dose-dependent relationship. Figure 3B ).

[0061] On the other hand, the cardiogenic activity of Codonopsis pilosula extract was evaluated using an embryo-like body (EB) formation model. The results showed that Codonopsis pilosula extract could promote the growth of beating EBs, consistent with results obtained in a stem cell differentiation model without EB formation (data not shown). These results suggest that Codonopsis pilosula extract contains potential components that can promote cardiogenic differentiation of undifferentiated EMG8 cells. Based on this, further in vivo animal model evaluations of Codonopsis pilosula extract were conducted.

[0062] Codonopsis pilosula extract improves cardiac function in rat model of myocardial infarction

[0063] The aforementioned experimental rats were bound to the left anterior descending artery (LAD) for one day, and then each rat was injected intraperitoneally with 10 mg of Codonopsis pilosula extract daily for 10 consecutive days. After 3 and 6 weeks, the fractional shortening (FS), fractional contraction area (FAC), and ventricular ejection fraction (EF) of the treated rats were monitored.

[0064] According to Table 1 below and Figures 4A-4CEchocardiographic data showed that cardiac function in rats did not significantly improve one week after administration of Codonopsis pilosula extract. However, after three weeks, the Codonopsis pilosula extract group (n=6) showed significant improvement in cardiac function, with FS at 40.8% (p<0.05), FAC at 59.1% (p<0.01), and EF at 40.2% (p<0.01), compared to the control group (n=6). After six weeks, FS improved by 22.6%, FAC by 35.3% (p<0.05), and EF by 29.6% (p<0.05). Although FAC and EF decreased in the Codonopsis pilosula extract group from week 3 to week 6, the damaged heart still showed significant functional improvement compared to the control group. These results confirm that Codonopsis pilosula extract can improve and promote cardiac function after infarction.

[0065] [Table 1]

[0066]

[0067] [Symbol Explanation]

[0068] none.

Claims

1. A method for promoting differentiation of stem cells, comprising: adding a Radix Codonopsis extract as a differentiation promoting agent to a medium in which the stem cells are cultured, wherein the stem cells differentiate into cells having cardiomyocyte-like activity, and wherein the Radix Codonopsis extract is a water extract and the Radix Codonopsis extract is extracted with water at 80-100 °C.

2. The method for promoting differentiation of stem cells according to claim 1, wherein the stem cells comprise embryonic stem cells or adult stem cells, and the embryonic stem cells are non-human mammalian embryonic stem cells.

3. Use of a Radix Codonopsis extract as a differentiation promoting agent for stem cells, wherein the Radix Codonopsis extract promotes differentiation of stem cells into cells having cardiomyocyte-like activity, and wherein the Radix Codonopsis extract is a water extract and the Radix Codonopsis extract is extracted with water at 80-100 °C.

4. The use of a Radix Codonopsis extract as a differentiation promoting agent for stem cells according to claim 3, wherein the stem cells comprise embryonic stem cells or adult stem cells, and the embryonic stem cells are non-human mammalian embryonic stem cells. ​

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