WO2018028370A1 - Cardiac stem cell and use thereof - Google Patents

Abstract

Disclosed are a cardiac stem cell and a use thereof, the cardiac stem cell expressing TNFR2. The cardiac stem cells can differentiate into cardiomyocytes and provide a new therapeutic strategy for treating human ischemic heart disease.

Description

Heart stem cell and use thereof Technical field

The invention relates to a cardiac stem cell and a use thereof, in particular to a cardiac stem cell expressing TNFR2 and the use thereof for preparing a medicament for treating human ischemic heart disease.

Background technique

Cardiovascular disease is one of the most serious diseases that threaten human life worldwide. Among them, acute myocardial infarction is the most serious disease in coronary heart disease, with high morbidity and mortality. Myocardial infarction causes a decrease in the number of irreversible functional myocardial cells, resulting in a decrease in myocardial contractile function. At present, the traditional treatment methods for myocardial infarction mainly include drug treatment, interventional therapy and surgical treatment. Although it can improve the symptoms of myocardial ischemia, it can not save the necrotic cardiomyocytes.

Stem cells are a kind of cells with self-renewal and multi-directional differentiation potential. They can replace damaged cardiomyocytes, construct new blood vessels, repair heart pumping function, and provide a new choice for the treatment of myocardial infarction. A growing body of evidence supports the presence of adult cardiac stem cells (CSCs) and is activated in an ischemic state, playing an important role in cardiac function repair.

Summary of the invention

It is an object of the present invention to provide a cardiac stem cell, and the present invention also provides the use of the cardiac stem cell, which provides a new therapeutic strategy for treating ischemic heart disease in humans.

To achieve the above object, the present invention adopts a technical solution of identifying a cardiac stem cell that expresses TNFR2.

At the same time, the present invention also provides the use of the cardiac stem cells in the preparation of cardiomyocytes.

In addition, the present invention also provides the use of the cardiac stem cells for the preparation of a medicament for treating myocardial infarction.

Further, the present invention provides a use of the cardiac stem cells for the preparation of a human ischemic heart disease.

The invention has the beneficial effects that the present invention provides a cardiac stem cell capable of expressing TNFR2, and a cardiac stem cell capable of differentiating into a cardiomyocyte, and providing a new treatment for ischemic heart disease in humans. The treatment strategy has great research significance.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an experimental technical roadmap of the present invention;

Figure 2 is a flow chart showing the method for obtaining iPSC-CSC by using the method of GiWi-heart stem cell differentiation; A.GiWi-heart stem cell differentiation method; B.QPCR detecting the expression of TNFR2, GATA4 and ISL1 in differentiated iPSC-CSC, vertical Coordinates indicate the percentage of target gene relative to internal reference (GAPDH) expression, abscissa indicates hESCs cell line and differentiated CPC cells; C. iPSC-CSC immunofluorescence staining images of TNFR2, NKX2.5 and GATA4 (10×); Immunofluorescence staining of TNFR2 and tropomyosin in iPSC-CSC (63×); immunofluorescence staining of NKX2.5 and tropomyosin in E.iPSC-CSC (63×); NKX2 in F.iPSC-CSC .5, immunofluorescence staining of TNFR2 and tropomyosin (63×); immunofluorescence staining of Ki67, TNFR2 and tropomyosin in GiPSC-CSC (63×);

Figure 3 shows the differentiation of CSC and mature CM under standard conditions; detection at the gene level (A) and protein level (B) by qRT-PCR and IB, respectively. Compared with the control group, OCT4 is only in humans. Expression in embryonic stem cells, whereas α-SA is only expressed in mature cardiomyocytes;

Figure 4 is a diagram showing the inhibition of cardiomyocyte differentiation by TNFR2 neutralizing antibody in Example 2 of the present invention. A-B: staining and quantification of NKx2.5 and cTnT positive cells. C: NKx2.5 mRNA expression is quantified;

Figure 5 is a diagram showing the effect of R2-TNF on cardiomyocyte differentiation; A, B: NKx2.5 and cTnT positive cells staining and quantification; and C: NKx2.5 mRNA expression quantification.

detailed description

The present invention will be further described in conjunction with the specific embodiments and the accompanying drawings, in which

The experimental technical route is shown in Figure 1.

The experimental method is specifically as follows:

The H1 hESCs cell line (US National Institutes of Health-registered as WA01) was transfected with a tetracycline-induced lentiviral vector or the control shRNA, TNFR2 or Bmx shRNA was expressed, and GFP was co-expressed. The induced shRNA system is used to antagonize factors known at each stage, such as Nkx2.5/Gata4 as a control. Instead, overexpressed TNFR2 or Bmx was constructed. The induced lentiviral shRNA system can down-regulate the target gene at a specific stage of differentiation. Transfection efficiency was detected using GFP co-expression and the transfected cells were specifically tracked. Down-regulated or up-regulated TNFR2 in hESCs was detected by qRT-PCR, IB, FACS.

Established methods to induce differentiation of hESCs. The standard differentiation step was adjusted in two ways: 1) TNFR2 neutralizing antibody or TNFR2 activator R2-TNF was added to the culture solution, and physiological saline, WT-TNF, and R1-TNF were used as controls. 2) The hypoxic chamber (5% CO ₂ , 0.5-1% O ₂ ) was used to simulate the ischemic environment and the myocardial differentiation was detected. The cells were replaced with fresh differentiation medium every 3 days and collected every three days until the cardiomyocytes were fully matured on day 30. Protein expression and TNFR2 signal, CSC marker (Nkx2.5/Gata4), differentiation marker (α-SA/cTnT) were detected, respectively.

The myofibrillar tissue was detected by transmission electron microscopy, and the beating region of the cultured cells was divided, fixed, and embedded with an electron microscope resin. Double staining with ultrathin sections of fluorescein acetate and citrate. Detection of myocardial activation drug isoproterenol (β1-adrenergic receptor agonist), Bay k8644 (Ca2+ channel activator), phenylephrine (α1-adrenergic receptor agonist), carbachol ( Cardiac chronotropic effects of muscarinic receptor agonists are evaluated for cardiomyocyte maturation. Single-cell randomized cells were used to detect the electrophysiological properties of mature cardiomyocytes, and whole-cell patch clamps were used to detect action potentials and calcium influx. The characteristic of terminal differentiation of cardiomyocytes is that cells stop proliferating and jump out of the cell cycle, so cell proliferation can also be detected by BrdU labeling. Neonatal rat brain cells were selected as controls for staining and contraction assays.

Example 1: Differentiation of hESCs and iPS into CSC and CMs cells

We used the Matrigel sandwich method established by Dr. Kamp’s group to culture and differentiate cells to increase cell production (Figure 2). The specific experimental method is as follows: hESC or iPS is monolayer cultured on Matrigel, the extracellular matrix is prepared, and then covered with matrigel. This method, like gastrulation, produces N-cadherin-positive mesenchymal cells and promotes EMT. Some growth factors (Activin A, BMP4, FGF) combined with Matrigel can be used to obtain high-purity (up to 98%) and high-number (up to 11CMs/hESC) cardiomyocytes from a variety of cells. Cardiomyocytes gradually matured in the culture medium for 30 days, and have myofilament expression and mitotic activity.

The experimental results are shown in Fig. 2: as shown by A in Fig. 2, on the 14th day of cell differentiation, the cells began to express cardiomyocyte characteristics, including spontaneous contraction and cardiac-related gene protein expression; As shown by B, we detected the cardiac markers GATA4 and Isl1 by qPCR, and the expression was significantly increased after differentiation. At the same time, as shown by the CG in Fig. 2, immunostaining showed that these cells were positive for TNFR2 and NKX2.5, and TNFR2/NKX2. .5 and TNFR2/Ki67 were positive.

In addition, we used similar experimental methods to observe TNFR2 expression and signaling in CSC production and differentiation. The results of the experiment are shown in Figure 3, using qRT-PCR and IB methods at the gene level (Figure 3 A) and protein level (Figure 3 B). Compared with the control group, OCT4 is only in human embryos. Expression in stem cells, while α-SA is only expressed in mature cardiomyocytes.

Example 2: Detection of TNFR2-Bmx signaling on differentiation of hESC/hiPSC-derived cardiomyocytes

The ultimate goal of the present invention is to use the TNFR2 activation strategy to treat ischemic heart disease in humans, but it is clear that we cannot use the human heart to define the role of the TNFR2-Bmx signaling pathway in eCSC, so we used TNFR2-positive exogenous cardiac stem cells-hESC/iPSC The source of CSC was studied to examine the effect of TNFR2-Bmx signaling on hESC/hiPSC-derived cardiomyocyte differentiation.

The experimental method is specifically as follows:

Established methods to induce differentiation of hESCs. The standard differentiation step was adjusted in two ways: 1) TNFR2 neutralizing antibody or TNFR2 activator R2-TNF was added to the culture solution, and physiological saline, WT-TNF, and R1-TNF were used as controls. 2) The hypoxic chamber (5% CO ₂ , 0.5-1% O ₂ ) was used to simulate the ischemic environment and the myocardial differentiation was detected. The cells were replaced with fresh differentiation medium every 3 days and collected every three days until the cardiomyocytes were fully matured on day 30. Protein expression and TNFR2 signal, CSC marker (Nkx2.5/Gata4), differentiation marker (α-SA/cTnT) were detected, respectively. The results are shown in Fig. 4 and Fig. 5. As can be seen from Fig. 4, the TNFR2 neutralizing antibody inhibits cardiomyocyte differentiation; as can be seen from Fig. 5, R2-TNF promotes cardiomyocyte differentiation.

It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention, although the present invention will be described in detail with reference to the preferred embodiments, The technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims (4)

A cardiac stem cell characterized in that the cardiac stem cell expresses TNFR2. The use of cardiac stem cells according to claim 1 for the preparation of cardiomyocytes. The use of a cardiac stem cell according to claim 1 for the manufacture of a medicament for the treatment of myocardial infarction. The use of cardiac stem cells according to claim 1 for the preparation of a human ischemic heart disease.

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