WO2014015777A1 - 体外诱导多能干细胞分化为心室肌细胞的方法 - Google Patents

体外诱导多能干细胞分化为心室肌细胞的方法 Download PDF

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WO2014015777A1
WO2014015777A1 PCT/CN2013/079811 CN2013079811W WO2014015777A1 WO 2014015777 A1 WO2014015777 A1 WO 2014015777A1 CN 2013079811 W CN2013079811 W CN 2013079811W WO 2014015777 A1 WO2014015777 A1 WO 2014015777A1
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stem cells
differentiation
cells
ventricular
signaling pathway
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PCT/CN2013/079811
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French (fr)
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马跃
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中国科学院生物物理研究所
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Priority to CN201380039458.1A priority Critical patent/CN104508121B/zh
Priority to US14/417,101 priority patent/US11339371B2/en
Priority to EP13823221.0A priority patent/EP2891712A4/en
Priority to JP2015523397A priority patent/JP6412868B2/ja
Priority to CA2886396A priority patent/CA2886396C/en
Priority to AU2013295940A priority patent/AU2013295940B2/en
Publication of WO2014015777A1 publication Critical patent/WO2014015777A1/zh
Priority to US17/746,583 priority patent/US20220340877A1/en

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Definitions

  • the present invention relates to the field of pluripotent stem cell differentiation and cell signal transduction, and more particularly to a method for inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro.
  • cardiomyocytes have the ability to divide and proliferate before birth, and this ability is rapidly reduced after birth. Cardiomyocytes in adults have little ability to divide and proliferate. In the case of cardiac tissue necrosis such as myocardial infarction, since the adult cardiomyocytes have lost the ability to proliferate and divide, and the necrotic tissue cannot be repaired by the regeneration of cardiomyocytes, the decline in cardiac function caused by such diseases is irreversible. Although the myocardial contractility can be increased by the use of drugs, and the heart's ability to pump blood can be increased, the increase in the burden of the heart may worsen the condition. Replacing necrotic cells with normal cardiomyocyte transplantation is one of the fundamental treatments for this type of heart disease. Because adult cardiomyocytes have almost lost the ability to divide and proliferate, finding the source of human cardiomyocytes has become the primary problem in the current regenerative medicine treatment of myocardial infarction.
  • Pluripotent stem cells such as embryonic stem cells (embryonic stem cells) 1, induced pluripotent stem cells (Induced Pluripotent Stem cells, iPSC) 2 '3, not only has a strong self-replication, but also differentiate into The potential of cardiomyocytes. Therefore, pluripotent stem cells such as embryonic stem cells are currently the most promising source of cardiomyocytes, and establishing efficient methods for the differentiation of cardiomyocytes is the key to obtaining cardiomyocytes.
  • pluripotent stem cells such as embryonic stem cells
  • the use of pluripotent stem cells, such as embryonic stem cells, to induce differentiation of cardiomyocytes generally involves two methods: one is to suspend embryonic stem cells to form an embryoid body and continue to differentiate into cardiomyocytes; another method is direct induction. Single-layer adherent embryonic stem cells under normal culture conditions are differentiated into cardiomyocytes. It has been reported in the literature that various cytokines have an effect of improving the efficiency of myocardial differentiation, and the amount and duration of action vary depending on the induced differentiation system.
  • Human pluripotent stem cell differentiated cardiomyocytes are mainly divided into three types: Nodal cell, Ventricular myocytes cell and Atrial myocyte cell.
  • the fully mature cardiomyocytes are based on their functional properties.
  • Can be divided into work Cardiomyocytes and autonomic pacing cardiomyocytes are two major categories.
  • the working myocardium is rich in myofibrils, conductive and excitatory, and performs contractile function. They are the main components of the atrium and ventricular wall, namely atrial myocytes and ventricular myocytes.
  • Autonomic pacing cardiomyocytes lose contractility, but they are as conductive and excitable as working cardiomyocytes. And they also have a unique self-discipline.
  • composition of intracellular myofibrils and the expression of ion channel proteins on the cell membrane of atrial myocytes, ventricular myocytes and pacemaker cells are significantly different, which results in a very high rhythm of action potential and contraction.
  • cardiomyocyte transplantation it is essential to transplant a single subtype of cardiomyocytes of the appropriate type and purity.
  • repairing ventricular tissue requires the transplantation of high-purity ventricular myocytes, which is related to whether it can be smoothly integrated into the recipient heart, whether it can really improve heart function, and whether it can reduce the side effects caused by arrhythmia caused by transplanted cells.
  • the transplanted cell type does not match or the purity of the cardiomyocytes is low, it will cause arrhythmia and damage the original cardiac function of the recipient. Because the left ventricle mainly bears the blood supply to the whole body, it has the largest volume, the thickest cell wall, and the strongest pumping ability, and the myocardial necrosis caused by myocardial infarction also occurs in this area. Thus, for the three types of cardiac cells, access to ventricular cells have greater significance five pairs of cell transplantation for myocardial infarction.
  • the heart is the most important organ of the human body. It is of great significance and application value to obtain a large number of human cardiomyocytes for the development of heart disease drugs and the safety evaluation of drugs. Since adult cardiomyocytes have almost no ability to divide and grow, they cannot be cultured and expanded in vitro. Therefore, no human cardiomyocytes can be used in a large number of experimental studies. Almost all experimental studies of cardiac toxicology experiments and cardiac drugs are performed using animal or primary isolated animal cardiomyocytes. However, due to the physiological differences between the animal heart and its cells and the human heart and cardiomyocytes, the accuracy of toxicological analysis of drugs using animal or animal cardiomyocytes is only about 60%, so the current drug toxicological analysis method remains to be determined. Further improvement and improvement.
  • Human cardiomyocytes obtained using stem cells or transdifferentiation can provide an effective new tool for cardiac toxicology analysis of drugs.
  • Human cardiomyocytes obtained by cell engineering can establish a toxicological analysis method of human heart at the cellular level, thereby reducing the use of animals in cardiac toxicology analysis and improving the accuracy of analysis, which is currently a hot spot in biomedical industry research. .
  • Due to the international regulations on drug registration (ICH S7B) it is required that the cardiac toxicology analysis of drugs must produce the effect of drugs on the ventricles, especially on ventricular heart rate. Therefore, in the cardiomyocytes obtained by cell engineering, ventricular myocytes have the most important significance for the development of drug toxicology analysis methods using human cardiomyocytes 6 .
  • the various methods for differentiating myocardium by stem cells have been reported to have the following defects:
  • the efficiency of inducing cardiomyocyte differentiation is not high, and the obtained cardiomyocytes are a mixed cell population of pacemaker cells, atrial myocytes, and ventricular myocytes, which cannot be realized.
  • Directional differentiation of different cardiomyocytes 4 .
  • CE Murry et al. used single-layer cultured human embryonic stem cells to directly induce differentiation of cardiomyocytes for heart transplantation in mice.
  • the average differentiation efficiency was only about 30%, that is, the last cardiomyocytes accounted for 30% of the total number of cells 7 .
  • After separation and purification by density gradient centrifugation a population of cardiomyocytes with a purity of about 80% can be obtained.
  • GM GM.
  • the method for differentiating induced pluripotent stem cells into ventricular myocytes in vitro is to maintain, expand and culture pluripotent stem cells in vitro, and to directly add to the medium in the middle of pluripotent stem cell differentiation. Or indirectly activate the substance of the Smadl/5/8 signaling pathway to differentiate into ventricular myocytes.
  • activating the Smadl/5/8 signaling pathway means phosphorylating one or more of the Smadl, Smad5, Smad8 proteins in the cytoplasm.
  • the period of differentiation of mesenchymal stem cells or cardiomyocyte precursor cells to cardiomyocytes in the middle stage of differentiation of pluripotent stem cells specifically refers to the expression of Brachyury (T) gene and/or Mespl gene from the cells, and differentiation into autonomously contracting myocardium. The period before the cell.
  • T Brachyury
  • the pluripotent stem cells are embryonic stem cells, induced pluripotent stem cells, embryonic germ cells or adult pluripotent stem cells, and the pluripotent stem cells are derived from human or breast-feeding. animal.
  • the substance and/or bone that can directly or indirectly activate the Smadl/5/8 signaling pathway Its final concentration is 0.01-1200 ng/mL.
  • a substance having a function of promoting cardiomyocyte differentiation is added to the medium.
  • the substance having the function of promoting cardiomyocyte differentiation is BMP4, basic fibroblast growth factor (bFGF), Activin A, Noggin, Dorsomorphin, 6-bromoindirubin-3'- ⁇ (6-bromoindirubin). At least one of -3'-oxime, BIO) and the like.
  • the final concentration of the added cell growth factor is 0.01-1200 ng/mL, and the final concentration of other small molecule compounds is 0.001-100 ⁇ .
  • the medium in the middle stage of pluripotent stem cell differentiation, is further added with A substance that inhibits the Wnt signaling pathway.
  • the substance having the Wnt signaling pathway is at least one of dickkopf homolog 1 (DKKl), IWP, IWR (inhibitors of Wntresponse), etc., and the final concentration of the added small molecule compound is 0.001-100 ⁇ .
  • the final concentration of DKKl is 0.01 - 1200 ng / mL.
  • an activator to which a retinoic acid receptor ⁇ (RARy) is added when the medium does not contain a retinoic acid or a substrate required for its synthesis Ii) When the medium contains retinoic acid or a substrate required for its synthesis, an antagonist of retinoic acid receptor a (RARa) and/or retinoic acid receptor ⁇ (RARJ3) is added thereto.
  • RARa retinoic acid receptor a
  • RARJ3 retinoic acid receptor ⁇
  • the present invention provides three sets of technical solutions:
  • cytokines that promote cardiomyocyte differentiation such as BMP4, bFGF, ActivinA, Noggin, etc.
  • BMP4 bFGF
  • ActivinA ActivinA
  • Noggin Noggin
  • small molecule compounds that inhibit the BMP pathway such as Dorsomorphin
  • Wnt3a small cells that can activate Wnt3a.
  • Molecular compounds such as BIO, CHIR99021, etc.;
  • a Wnt signaling pathway is inhibited by adding a protein growth factor or a small molecule compound such as DKK1, IWP or IWR, which inhibits the Wnt pathway, and BMP2 and/or BMP4 can be activated at the same time.
  • Smadl/5/8 phosphorylated signaling molecules are directed to induce cell differentiation into ventricular myocytes.
  • a Wnt signaling pathway is inhibited by adding a protein growth factor or a small molecule compound such as DKK1, IWP or IWR, which inhibits the Wnt pathway, and simultaneously stimulates the expression and secretion of the cell to activate Smadl. /5/8 phosphorylation (1) Suspension culture or monolayer culture of substantially undifferentiated pluripotent stem cells;
  • cytokines that promote cardiomyocyte differentiation such as BMP4, bFGF, Activin A, Noggin, etc.
  • BMP4 cytokines that promote cardiomyocyte differentiation
  • bFGF Activin A
  • Noggin etc.
  • small molecule compounds that inhibit the BMP pathway such as Dorsomorphin
  • Wnt3a small molecule compounds, such as BIO;
  • a Wnt signaling pathway is inhibited by adding a protein growth factor or a small molecule compound such as DKK1, IWP or IWR, which inhibits the Wnt pathway, and at the same time, contains retinoic acid or a synthetic base thereof.
  • An antagonist of the retinoic acid receptor RARa and/or RARP (such as an antagonist of RARa Ro41-5253 and an antagonist of RARJ3 LE135) is added to the medium.
  • stem cell-differentiated cardiomyocytes can be induced into cardiomyocytes predominantly ventricular myocytes.
  • the differentiation process was changed from the 14th day to the factor-free medium every 3 days; after 60-90 days of culture, the action potential of cardiomyocytes was detected by patch clamp technique, calcium signal recording, MLC-2v and cTNT double staining Differentiated ventricular myocytes and non-ventricular myocytes (including atrial myocytes and pacemaker cells) were determined by flow cytometry and the like.
  • the present invention further provides the use of ventricular myocytes prepared by the above method for screening cardiac toxicological tests for treating cardiac drugs and drugs.
  • the invention further provides the use of ventricular myocytes prepared by the above method for stem cell therapy of a damaged heart.
  • the present invention also provides a method of promoting differentiation of stem cells into ventricular myocytes, the method comprising activating a Smadl/5/8 signaling pathway in mesoderm cells; wherein said mesodermal cells are differentiated from stem cells.
  • the stem cells are a Totipotent stem cell, a Pluripotent stem cell, a Multipotent stem cell, an Oligopotent stem cell or a unipotent stem cell (Unipotent). Stem cell ).
  • the stem cells are embryonic stem cells, induced pluripotent stem cells, fetal stem cells or adult stem cells.
  • the stem cells are mammalian stem cells.
  • the mammalian stem cell is a human stem cell.
  • the stem cells are human embryonic stem cells or induced human pluripotent stem cells.
  • the stem cells are differentiated into mesoderm cells by contacting the undifferentiated stem cells with at least one of the following substances: bFGF, bone morphogenetic protein 2 (BMP2), BMP4, Activin A, BMP Antagonists, BMP signaling pathway inhibitors, Wnt3a signaling pathway activators, etc.
  • the BMP antagonist is a BMP4 antagonist.
  • the BMP antagonist is Noggin.
  • the BMP signaling pathway inhibitor is a small molecule BMP signaling pathway inhibitor.
  • the small molecule BMP signaling pathway inhibitor is Dorsomorphin.
  • the Wnt3a signaling pathway activator is a small molecule Wnt3a signaling pathway activator.
  • the small molecule Wnt3a signaling pathway activator is an ATP-competitive inhibitor of GSK-3a/p.
  • the ATP-competitive inhibitor of the GSK-301/ ⁇ is a cell-permeable bw-indolo (indirubin) compound.
  • the cell is infiltrated with a biguanide (Beiyuhong) compound as BIO.
  • a biguanide Beiyuhong
  • the final concentration of the added growth factor is 0.01-1200 ng/mL, and the final of other substances The concentration is 0.001-100 ⁇ .
  • stem cells are contacted with ⁇ 2 and/or ⁇ 4 to activate the Smadl/5/8 signaling pathway.
  • the final concentration of BMP2 and I or BMP4 used is about 0.01 to 1200 ng/mL.
  • the Smadl/5/8 signaling pathway is activated by culturing stem cells in a medium containing no retinoic acid or a precursor thereof, and contacting the stem cells with a retinoic acid receptor ⁇ (RARy) activator.
  • the retinoic acid precursor is vitamin A.
  • the RARy activator is BMS961, Palovarotene or purchased from
  • the RARy activator is used at a final concentration of about 0.001 to 100 ⁇ M.
  • stem cells are contacted with a retinoic acid receptor a (RARa) and/or a retinoic acid receptor beta (RAR) antagonist to activate the Smadl/5/8 signaling pathway.
  • RARa retinoic acid receptor a
  • RAR retinoic acid receptor beta
  • the RARa antagonist is Ro41-5253, BMS 195614 or ER50891, and the RARJ antagonist is LE135 or the like.
  • the RARa and I or RARP antagonists are used at a final concentration of 0.001 to 100 ⁇ M.
  • the Wnt inhibitor is at least one of DKK1, IWP, Wntresponse inhibitor, and the like.
  • the Wnt inhibitor DKK1 is used at a final concentration of 0.01 to 1200 ng/mL, and the other inhibitors have a final concentration of 0.001 to 100 ⁇ M.
  • the present invention also provides ventricular myocytes prepared according to the above method.
  • ventricular myocytes Expression levels or ratios of ventricular myocytes, ventricular-specific expression genes, embryonic ventricular-like action potentials (AP), and/or representative features of ventricular myocytes, Ca 2+ sparks (Ca 2+ spark ) Raise.
  • the ventricular-specific expression genes are IRX-4 and/or MLC-2v.
  • the invention also provides a composition comprising stem cells that have been differentiated into mesodermal cells and treated with an exogenous substance capable of activating the Smadl/5/8 signaling pathway in stem cells.
  • the exogenous substance capable of activating the Smadl/5/8 signaling pathway in stem cells is BMP2 and/or BMP4.
  • the aforementioned composition which is capable of activating the Smadl/5/8 signaling pathway in stem cells
  • the source material is a RARy activator.
  • the exogenous substance capable of activating the Smadl/5/8 signaling pathway in stem cells is an RARa and/or RARJ antagonist.
  • the present invention also provides a method for producing ventricular myocytes from stem cells, the method comprising: 1) contacting stem cells with bFGF and BMP4 to induce cell differentiation; 2) contacting stem cells treated with bFGF and BMP4 with Activin A to form mesoderm 3) The stem cells that have differentiated into mesoderm cells are contacted with Noggin to increase the efficiency of stem cells to differentiate into cardiomyocytes; 4) Activate the Smadl/5/8 signaling pathway in stem cells treated with Noggin to promote ventricular muscle The formation of cells; 5) contacting the stem cells treated with Noggin with at least one of the following substances to differentiate the stem cells into ventricular myocytes; the substance is at least one of DKK1, IWP, IWR, and the like.
  • the Smadl/5/8 signaling pathway is activated by contacting the stem cells with BMP2 and/or BMP4.
  • the stem cells are cultured in a medium containing no retinoic acid or its precursor vitamin A, and the stem cells are contacted with the RARy activator to increase the expression level of BMP2/4 in the cultured cells, thereby activating Smadl/5. /8 signal path.
  • the Smadl/5/8 signaling pathway is activated by contacting the stem cells with RARa and/or RARP antagonists.
  • the present invention also provides ventricular myocytes prepared according to the above method.
  • the invention also provides a pharmaceutical composition for treating heart damage or heart disease comprising an effective amount of the aforementioned ventricular myocytes and a pharmaceutically acceptable carrier or excipient.
  • the invention also provides a method of treating cardiac damage or heart disease, the method comprising administering to an individual in need of such treatment an effective amount of the above pharmaceutical composition.
  • the individual is a human.
  • the foregoing method is used for stem cell treatment of cardiac damage.
  • the present invention also provides the use of the aforementioned ventricular myocytes for the preparation of a medicament for treating and/or preventing cardiac damage or heart disease.
  • the present invention also provides the use of the aforementioned ventricular myocytes for screening and/or developing a medicament for treating and/or preventing heart damage or heart disease.
  • the invention also provides the use of the aforementioned ventricular myocytes for cardiac toxicology testing of drugs.
  • the invention further provides a method for identifying a ventricular myocyte regulatory factor, the method comprising: 1) contacting the ventricular myocytes with a candidate regulatory factor, and determining the effect of the candidate regulatory factor on ventricular myocyte function; The function of the ventricular myocytes contacted by the candidate regulatory factor; if the function of the ventricular myocytes contacting the candidate regulatory factor is different than the function of the ventricular myocytes not contacted by the candidate regulatory factor, determining the candidate regulatory factor is A functional regulator of ventricular myocytes.
  • the method for inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro by the present invention can activate Smadl/5/ by direct induction of stem cells in the middle stage of cardiac differentiation of stem cells by directly adding BMP2 and/or BMP4 to the culture system.
  • the substance of the 8 signaling pathway can differentiate stem cells into cardiomyocytes mainly composed of ventricular myocytes.
  • the addition of substances such as BMP2 and or BMP4 that activate the Smad 1/5/8 signaling pathway can effectively inhibit myocardial preconditioning in the presence of retinoic acid or a precursor (such as vitamin A) required for its synthesis. Somatic cells differentiate into atrial myocytes and induce differentiation of ventricular myocytes.
  • the ratio of the central atrial myocytes of the differentiated cardiomyocytes decreases as the concentration of BMP4 increases.
  • the ratio of differentiated cardiomyocytes to the central chamber muscle cells increases. If only BMP2/4 is added in the middle stage of stem cell myocardial differentiation, and retinoic acid or a precursor required for its synthesis is removed in the medium, the myocardial precursor cells can be efficiently differentiated into ventricular myocytes.
  • the activator of RARa or RARP which is added with retinoic acid in the medium, can effectively inhibit the differentiation of iota muscle cells, which is expressed by the expression of the early marker gene IRX-4 in ventricular myocytes. inhibition.
  • the addition of RARy's activator can effectively induce stem cells to differentiate into ventricular myocytes.
  • the addition of antagonists of RARa and RARJ to a medium containing vitamin A or retinoic acid can also effectively increase the expression level of IRX-4 and induce differentiation of stem cells into ventricular myocytes.
  • the BMP and Smadl/5/8 pathways positively regulate the differentiation of ventricular myocytes in the differentiation of stem cells, which lays a foundation for the subsequent utilization of stem cell differentiation to obtain high-purity ventricular myocytes.
  • the in vitro induced pluripotent stem cells provided by the present invention to differentiate into ventricular myocytes
  • the method can successfully obtain high-purity, biologically active and functional ventricular myocytes without any purification step, which not only lays a solid theoretical foundation for revealing the regulatory mechanism of myocardial precursor cells to ventricular myocytes differentiation. Moreover, it is of great significance for cell transplantation for myocardial infarction and drug research and development using cardiomyocytes differentiated from human stem cells.
  • Figure 1 shows the expression of BMP signaling pathway and the expression of ventricular myocyte specific marker gene IRX-4 in the middle stage of stem cell differentiation.
  • A is the detection of BMP2 by RT-PCR on the 5th and 6th day of stem cell myocardial differentiation.
  • BMP4 and its receptor expression B is Westen blot detection of downstream signaling of BMP pathway Smadl/5/8 is phosphorylated, P-Smadl/5/8 is phosphorylated Smadl/5/8 protein molecule, T-Smadl/5/8 is the total Smadl/5/8 protein molecule, ⁇ -actin is the internal reference protein;
  • C column histogram indicates the quantitative RT-PCR detection of IRX-4 gene expression level on the 14th day of differentiation.
  • the results showed that the expression level of IRX-4 when ⁇ retinoic acid and 200 ng/mL of BMP4 were added to the medium in different time periods, and the line label showed the efficiency of stem cell myocardial differentiation under the corresponding conditions; N stands for Noggin, B stands for BMP4, NVa is a differentiated cell treated with medium without vitamin A, RA stands for retinoic acid, the number indicates the concentration, and the concentration of BMP4 is ng/mL.
  • the result of quantitative PCR is compared with the expression of GADPH. of Value.
  • Figure 2 shows the expression level of early specific expression gene IRX-4 of ventricular myocytes in different treated differentiated stem cells on day 14 of differentiation by quantitative PCR; wherein A shows IRX-4 after adding different concentrations of BMP4 to the medium.
  • the expression increased with the increase of BMP concentration, but decreased after the addition of BMP antagonist Noggin; B is the addition of different doses of Noggin in the medium containing no retinoic acid precursor vitamin A can effectively reduce IRX
  • the expression level of -4 is C; in the presence of ⁇ retinoic acid, the expression level of IRX-4 increases with the concentration of ⁇ 4 added; D shows that ⁇ 4 enhances the expression of IRX-4 in the presence of retinoic acid
  • N represents Noggin
  • B represents BMP4
  • NVa is a differentiated cell treated without vitamin A medium
  • RA represents retinoic acid
  • the concentration is expressed in units of ng/ mL.
  • the results of quantitative PCR are relative values
  • Figure 3 is a quantitative RT-PCR analysis of the expression level of the IRX-4 gene on day 14 of differentiation.
  • the results showed that other members of the BMP family treated stem cells on day 5-8 of stem cell cardiomyocyte differentiation to effectively antagonize the inhibitory effect of retinoic acid on the early expression of gene IRX-4 in ventricular myocytes, and the antagonism was accompanied by the addition of BMP family growth factors.
  • the dose increases and increases.
  • RA stands for retinoic acid, the number indicates the concentration of growth factor, the unit is ng/mL, and the concentration of retinoic acid is 1 ⁇ .
  • the result of quantitative PCR is a relative value compared to the amount of GADPH expression.
  • Figure 4 shows the expression of the specific expression gene MLC-2v when stem cells treated with retinoic acid, ⁇ 4 and Noggin differentiate into mature ventricular myocytes.
  • A is a cardiomyocyte differentiated by retinoic acid and BMP4-induced stem cells by Western blot. Expression of MLC-2v after 90 days of differentiation; B is the result of immunofluorescence staining of cTNT and MLC-2v double staining of cardiomyocytes after 90 days of differentiation treated with retinoic acid, BMP4 and Noggin; wherein B represents BMP4, and NVa is vitamin-free.
  • a medium-treated differentiated cells, RA indicates ⁇ retinoic acid, and the number indicates the concentration in ng/mL.
  • FIG. 5 is a result of confocal image scanning and action potential recording of calcium activity of differentiated cardiomyocytes and classification of differentiated cardiomyocytes using various cardiomyocyte-specific calcium patterns;
  • A shows ventricular myoelectric action potential (ventricular) -like action potential) Cardiomyocytes have calcium sparks (Ca 2+ Spark), cardiomyocytes with atrial-like action potential have calcium variability (Ca 2+ Transient) characteristics, and have pacing Cardiomyocytes with Nodal-like action potential have the characteristics of Ca 2+ Oscillation;
  • B is the cardiomyocyte obtained from different treatment groups by using the calcium signal pattern of different subtypes of cardiomyocytes obtained in A.
  • the cardiomyocytes in each group obtained by classification have the ratio of calcium spark, calcium transient and calcium oscillation, and the ordinate indicates the ratio of three different types of calcium activity in the detected cardiomyocytes; RA indicates the retinoic acid of ⁇ , ⁇ indicates ⁇ 4, NVa indicates no vitamin ⁇ medium, N indicates Noggin, and the number indicates concentration in ng/mL.
  • Figure 6 is a quantitative RT-PCR assay for the expression level of BMP2 in cells treated with RARy activator on day 6 of cell differentiation. Among them, the result of quantitative PCR is the relative value compared with the amount of GADPH expression. NVa means no vitamin A medium.
  • Fig. 7 is a quantitative RT-PCR analysis of ventricular muscle specificity on the 14th day of differentiation after adding a different retinoic acid receptor activator or inhibitor to a medium containing no vitamin A in the middle stage of stem cell myocardial differentiation.
  • the RAR-pan antagonist is BMS493.
  • the result of quantitative PCR is a relative value compared with the amount of cTNT expression.
  • Figure 8 shows the identification of single cell action sites and mature ventricular myocyte specific marker gene MLC-2v in different induced differentiation of cardiomyocytes.
  • A is the ratio of cells with action potentials of atrial, ventricular and pacemaker cells in cardiomyocytes differentiated by different induction conditions (n>30).
  • B is a flow cytometry analyzer for detecting the ratio of MLC-2v cells expressed in cardiomyocytes (cTNT positive cells) in a 90-day differentiated cell population treated with different conditions.
  • RA stands for ⁇ of retinoic acid, ⁇ means ⁇ 4, NVa means no vitamin ⁇ medium, N means Noggin, and the number indicates concentration in ng/mL. The concentration of RARy was 0.1 ⁇ .
  • Fig. 9 is a view showing the process of inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro according to Example 2 of the present invention.
  • Fig. 10 is a view showing the process of inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro in Example 3 of the present invention.
  • Figure 11 is a diagram showing the process of inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro in Example 4 of the present invention.
  • Figure 12 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with retinoic acid.
  • Figure 13 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with retinoic acid and 200 ng/mL BMP4.
  • Figure 14 shows the results of immunofluorescence co-staining of cTnT and MLC-2v after differentiation of cardiomyocytes treated with vitamin A-free medium and 1200 ng/mL Noggin.
  • Fig. 15 shows the results of immunofluorescence co-staining of the differentiated cardiomyocytes finally obtained in Example 2 as cTnT and MLC-2v.
  • Figure 16 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with vitamin A-free medium.
  • Fig. 17 shows the results of immunofluorescence co-staining of the differentiated cardiomyocytes finally obtained in Example 3 as cTnT and MLC-2v.
  • the human embryonic stem cell line H7 used in the following examples was purchased from WiCell Research Institute, USA; B27 medium supplement and RPMI 1640 medium, purchased from Invitrogen; Activin A, bFGF, DKK1, BMP4 and Noggin were purchased from R&D systems.
  • IRX-4 is known to be a marker gene for early expression during ventricular myocyte differentiation.
  • the present invention further investigates the role of the BMP signaling pathway in the determination of myocardial subtypes by detecting the expression level of IRX-4.
  • Noggin a BMP2/4 inhibitor
  • the addition of Noggin to stem cells differentiated from vitamin A-free medium in the middle of stem cell differentiation can effectively reduce the expression of IRX-4 in differentiated cells, and the expression level of IRX-4 is The amount of Noggin was increased (300 ng/mL, 600 ng/mL, and 1200 ng/mL) and decreased, as shown in Figure 2.
  • 3 Experiments have shown that the addition of retinoic acid in the middle stage of stem cell myocardial differentiation can inhibit the expression of ventricular myocardial specific gene IRX-4.
  • IRX-4 Other members of the BMP family are also able to antagonize the inhibitory effect of retinoic acid on IRX-4.
  • Members of the BMP family have similar functions.
  • quantitative RT-PCR experiments have shown that in the middle of stem cell myocardial differentiation, other members of the BMP family can be improved to varying degrees in the presence of ⁇ retinoic acid.
  • the expression level of IRX-4, and the expression level of IRX-4 increased as the concentration of the growth factor added increased.
  • BMP signaling pathway can effectively increase the expression of IRX-4 in differentiated cardiomyocytes, thus revealing that BMP signaling pathway is involved in stem cell differentiation.
  • Early differentiation of central chamber muscle cells promotes differentiation of ventricular myocytes.
  • the present invention performed confocal image scanning studies on calcium activity of cardiomyocytes differentiated for 60-90 days. There is a clear distinction between ventricular myocytes and calcium activity in atrial myocytes and pacemaker cells.
  • the imaging activity of calcium activity in ventricular myocytes is faster, called “Ca + sparks”; and the imaging of calcium activity in atrial myocytes shows that the frequency is slower and the signal is larger, called “Ca + transients”; Imaging of pacemaker cell calcium activity shows significant periodicity, known as "calcium oscillation.”
  • Ca + sparks The imaging activity of calcium activity in ventricular myocytes
  • atrial myocytes shows that the frequency is slower and the signal is larger, called “Ca + transients”
  • Imaging of pacemaker cell calcium activity shows significant periodicity, known as "calcium oscillation.”
  • the calcium activity of 20 cells with ventricular muscle action potential recorded in patch clamp was all calcium spark; 20 lesions with atrial muscle action potential recorded in patch clamp All cells had calcium transients; all 20 cells with pacemaker cell action potential recorded in patch clamps had calcium oscillations. Therefore, imaging of cardiomyocyte calcium activity can effectively distinguish ventricular myocytes from atrial myocytes and pacemaker cells.
  • IRX-4 is an important specific expression gene for ventricular myocyte differentiation.
  • MLC-2v myosin light chain 2 gene. Therefore, the present invention also examined the expression level of MLC-2v in differentiated cells cultured for 90 days treated with different growth factors. Western blot showed that the protein expression level of MLC-2v increased with the increase of the concentration of BMP4 in the cells differentiated for 90 days (Fig. 4); the present invention also detected the cells differentiated by 90 days using flow cytometry. The ratio of ventricular myocytes expressing MLC-2v to the differentiated cardiomyocytes (cells expressing cTNT).
  • the addition of BMP treatment to the differentiated cells treated with retinoic acid can effectively increase the ratio of expression of MLC-2v cells in cardiomyocytes, and express MLC- under the condition of treatment with BMP4 alone.
  • the ratio of 2v cells reached the highest.
  • the most classical method for identifying cardiomyocyte subtypes is to measure the action sites of cardiomyocytes. By detecting the ratio of action potentials of atrial, ventricular myocytes, and pacemaker cells in differentiated cardiomyocytes treated with retinoic acid and different doses of BMP4 (Fig. 8), in retinoic acid-treated differentiated cells, with the addition With an increase in the BMP4 dose, the ratio of cells with ventricular muscle action sites increased significantly. More than 90% of the cardiomyocytes in the differentiated cells to which BMP4 is added alone have ventricular myoelectric action potential, that is, more than 90% of the cardiomyocytes are ventricular myocytes.
  • the flow cytometry test proved (Fig. 8) that the ratio of ventricular myocytes expressing MLC-2v in the RARy-induced 90-day differentiated cardiomyocytes to the differentiated cardiomyocytes (cTNT-expressing cells) can reach 80%. the above.
  • the electrophysiological action potentials of RARy-induced 90-day-differentiated cardiomyocytes were identified, indicating that 92% of the cardiomyocytes in the differentiated cells have ventricular myoelectric action potential and are ventricular myocytes.
  • Example 2 Differentiation of pluripotent stem cells into ventricular myocytes in vitro (Technical Protocol 1) Human embryonic stem cells H7 were plated in gelatin-containing gelatin dishes, and RPMI1640 medium containing B27 (1 ⁇ concentration) was added at 37 Incubate in a °C C0 2 cell culture incubator. The process of myocardial differentiation was as shown in Fig. 9. From day 0 to day 3, Activin A (10 ng/mL), BMP4 (6 ng/mL) and bFGF (6 ng/mL) were added to the medium. The medium was changed at the end of the third day while the inhibitor of No. 2, OOng/mL of BMP2/4 was added.
  • the medium was changed to B27-containing RPMI 1640 medium without the addition of vitamin A.
  • Wnt3a inhibitors DKK1 (300 ng/mL) and BMP4 (10 ng/mL) were added to the medium.
  • the medium was changed to a medium containing only 300 ng/mL DKKl, and the medium was changed at the end of the 10th day of differentiation without adding any growth factor.
  • B27-added RPMI1640 medium was replaced every 3 days thereafter. A large number of beating cardiomyocytes can be observed at 14 days.
  • the program flow is shown in Figure 9.
  • Example 3 Differentiation of pluripotent stem cells into ventricular myocytes in vitro (Technical Protocol 2) Human embryonic stem cells H7 were plated in gelatin-containing gelatin dishes, and RPMI1640 medium containing B27 (1X concentration) was added at 37 Incubate in a °C C0 2 cell culture incubator. The process of myocardial differentiation was as shown in Fig. 10. From day 0 to day 3, Activin A (10 ng/mL), BMP4 (6 ng/mL) and bFGF (6 ng/mL) were added to the medium. At the end of day 3, the medium was changed and BMP2/4 inhibitor Noggin (300 ng/mL) was added.
  • the medium was changed to B27-containing RPMI 1640 medium without the addition of vitamin A.
  • Wnt3a inhibitor DKK1 300ng/mL
  • retinoic acid receptor RARy activator BMS961 ⁇ . ⁇ ⁇ , purchased from Wnt3a were added to the medium. Tocris).
  • the medium was changed to a medium containing only 300 ng/mL DKKl, and the medium was changed at the end of the 10th day of differentiation without adding any growth factor.
  • B27-added RPMI1640 medium was replaced every 3 days thereafter. A large number of beating cardiomyocytes were observed at 14 days.
  • the program flow is shown in Figure 10.
  • Example 4 Differentiation of pluripotent stem cells into ventricular myocytes in vitro (Technical Protocol 3) Human embryonic stem cells H7 were plated in gelatin-containing gelatin dishes, and RPMI1640 medium containing B27 (1X concentration) was added at 37 Incubate in a °C C0 2 cell culture incubator. The process of myocardial differentiation was as shown in Fig. 11. From day 0 to day 3, Activin A (10 ng/mL), BMP4 (6 ng/mL) and bFGF (6 ng/mL) were added to the medium. At the end of day 3, the medium was changed and BMP2/4 inhibitor Noggin (300 ng/mL) was added.
  • B27-containing RPMI 1640 medium containing vitamin A was used.
  • Wnt3a inhibitor DKKl 300 ng/mL
  • retinoic acid receptor RARa and ⁇ antagonists BMS 195614 and LE135 were added to the medium.
  • the medium was changed to a medium containing only 300 ng/mL DKKl, and the medium was changed at the end of the 10th day of differentiation without adding any growth factor.
  • B27-added RPMI1640 medium was replaced every 3 days thereafter. A large number of beating cardiomyocytes were observed at 14 days.
  • the program flow is shown in Figure 11.
  • Figure 12 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with retinoic acid.
  • Figure 13 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with retinoic acid and 200 ng/mL BMP4.
  • Figure 14 shows the results of immunofluorescence co-staining of cTnT and MLC-2v after differentiation of cardiomyocytes treated with vitamin A-free medium and 1200 ng/mL Noggin.
  • Fig. 15 shows the results of immunofluorescence co-staining of the differentiated cardiomyocytes finally obtained in Example 2 as cTnT and MLC-2v.
  • Figure 16 shows the results of immunofluorescence co-staining of cTnT and MLC-2v differentiated cardiomyocytes treated with vitamin A-free medium.
  • Fig. 17 shows the results of immunofluorescence co-staining of the differentiated cardiomyocytes finally obtained in Example 3 as cTnT and MLC-2v.
  • the cells marked with “*” are non-ventricular myocytes, with " ⁇ "
  • the labeled cells are ventricular myocytes expressing MLC-2v.
  • the final concentration of each additive in the medium involved in the above examples is such that the final concentration of the growth factor is 0.01-1200 ng/mL, and the concentration of the small molecule compound is 0.001-100 ⁇ .
  • the ventricular myocytes with biological activity and function are successfully obtained, which not only reveals the regulatory mechanism of differentiation of myocardial precursor cells into ventricular myocytes, but also differentiates human ventricular myocytes to treat myocardial cells by cell transplantation. Infarction and cardiac toxicology analysis and the development of cardiac related drugs have a wide range of applications.

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Abstract

本发明提供体外诱导多能干细胞分化为心室肌细胞的方法，其是通过在体外维持、扩增、培养多能干细胞，在多能干细胞心肌分化的中期即由中胚层细胞或心肌前体细胞向心肌细胞分化的时期，向培养基中加入可直接或间接激活Smad1/5/8信号通路的物质，使干细胞定向分化为心室肌细胞。利用本发明方法，成功获得了具有生物学活性及功能的心室肌细胞，不仅揭示了心肌前体细胞向心室肌细胞分化过程中的调节机制，而且分化得到的人心室肌细胞对细胞移植治疗心肌梗死和心脏毒理分析及心脏相关药物的研发有广泛的应用。

Description

体外诱导多能干细胞分化为心室肌细胞的方法
技术领域
本发明涉及多能干细胞分化及细胞信号转导领域， 具体地说， 涉 及体外诱导多能干细胞分化为心室肌细胞的方法。
背景技术
对于人类和哺乳动物而言，心肌细胞在出生前具有分裂增殖的能 力， 而出生以后， 这一能力迅速降低。 成年人的心肌细胞几乎不具备 分裂增殖的能力。 在发生诸如心肌梗塞等心脏组织坏死性疾病时， 由 于成体心肌细胞已失去增殖分裂的能力，不能通过心肌细胞的再生来 修复坏死的组织， 所以这类疾病引起的心脏功能衰退是不可逆转的。 虽然可以通过药物的使用增加心肌收缩力， 提高心脏的泵血能力， 但 心脏负担的加重反而可能使病情恶化。用正常的心肌细胞移植替换已 经坏死的细胞是从根本上治疗这类心脏病的方法之一。因为成体心肌 细胞几乎已失去分裂增殖能力，寻找人心肌细胞的来源成为目前再生 医学治疗心肌梗塞首要解决的问题。
能性干细胞（ Pluripotent stem cells ),如胚胎干细胞 (embryonic stem cells)1 , 诱导的多能干细胞（Induced Pluripotent Stem cells, iPSC ) 2' 3, 不但具有较强的自我复制能力,而且还具有分化成心肌细胞的潜能。 因 此， 例如胚胎干细胞的多能干细胞是目前最具潜力的心肌细胞来源， 而 建立高效的， 心肌细胞定向分化方法是获得心肌细胞的关键。
利用多能干细胞如胚胎干细胞，诱导分化心肌细胞一般包括两种 方法：一种是悬浮培养胚胎干细胞，使其形成胚状体（embryoid body ) 进而继续分化成心肌细胞；另一种方法是直接诱导一般培养条件下的 单层贴壁胚胎干细胞， 使其向心肌细胞分化。 已有文献报道多种细胞 因子具有提高心肌分化效率的作用，其用量及作用时间因不同的诱导 分化体系而异。
人的多能干细胞分化的心肌细胞主要分为三类：起搏细胞（Nodal cell ), 心室肌细胞 （ Ventricular myocytes cell ) 和心房肌细胞 （Atrial myocyte cell ) 完全成熟的心肌细胞根据其功能属性又可分为工作 心肌细胞和自律性的起搏心肌细胞两大类。工作心肌细胞的肌原纤维 丰富， 具有传导性和兴奋性， 执行收缩功能。 它们是心房和心室壁的 主要构成部分， 即心房肌细胞和心室肌细胞。 具有自律性的起搏心肌 细胞丧失了收缩性， 但它们和工作心肌细胞一样具有传导性和兴奋 性。 而且它们还具有独特的自律性。 心房肌细胞、 心室肌细胞和起搏 细胞这三类细胞的胞内肌原纤维的组成以及细胞膜上的离子通道蛋 白表达有明显区别，这也就造成它们的动作电位和收缩的节律性有很 大差别。 对于心肌细胞移植而言， 至关重要的是要移植适当种类且纯 度较高的单一亚型的心肌细胞。 例如， 修复心室组织需要移植高纯度 的心室肌细胞， 这关系到其能否顺利整合到受体心脏、 能否真正提高 心脏功能和是否能够减少移植细胞所带来的心律不齐等副作用。如果 移植细胞种类不匹配或纯度不高的心肌细胞， 会造成心律不齐， 损害 受体的原有心脏功能。 由于左心室主要负担全身的血液供给， 其体积 最大， 细胞壁也最厚， 泵血能力最强， 而心肌梗死导致的心肌细胞坏 死也多发于该区域。 因此， 对于三类心肌细胞来说， 获得心室肌细胞 对细胞移植治疗心肌梗死具有更为重要的意义 5。
心脏是人体最重要的器官，获取大量的人心肌细胞对心脏病药物 的研制和药物的安全性评估具有重要意义和应用价值。由于成年人的 心肌细胞几乎没有分裂生长能力， 不能进行体外培养扩增。 因此， 目 前没有人的心肌细胞可以大量地用于实验研究。几乎所有的心脏毒理 实验和心脏病药物的实验研究都是利用动物或原代分离的动物心肌 细胞来完成。 但是， 由于动物心脏及其细胞与人心脏及心肌细胞的生 理差异，利用动物或动物的心肌细胞来进行药物的毒理分析的准确率 只有 60%左右， 因此目前的药物毒理分析方法还有待进一步改进和提 高。利用干细胞或转分化而获得的人的心肌细胞可以为药物的心脏毒 理分析提供一种有效的新工具。利用细胞工程获取的人的心肌细胞可 以在细胞水平上建立人心脏的毒理分析方法，从而减少心脏毒理分析 中动物的使用， 提高分析的准确率， 这是目前生物医药工业研究的一 个热点。 由于国际上药物注册的相关规定条款（ICH S7B ) 要求药物 的心脏毒理分析必须出具药物对心室， 特别是对心室心率影响的数 据， 因此， 利用细胞工程获取的心肌细胞中， 心室肌细胞对发展利用 人心肌细胞药物毒理分析方法具有最为重要的意义 6。
综上，无论是利用干细胞分化的心肌细胞治疗进行细胞移植治疗 心肌梗死， 还是利用获取的心肌细胞进行药物的心脏毒理分析， 都需 要获得高纯度的人的心室肌细胞。 因此， 揭示心肌前体细胞向心室肌 细胞分化过程中的调节机制不仅对高效诱导获得心室肌细胞具有重 要的意义，而且还对细胞移植治疗心肌梗死和利用人干细胞分化的心 肌细胞进行药物研发具有重要意义。
现已报道的利用干细胞分化心肌的各种方法主要存在以下缺陷： 诱导心肌细胞分化的效率不高， 且得到的心肌细胞是起搏细胞、 心房 肌细胞和心室肌细胞的混合细胞群体， 不能实现不同心肌细胞的定向 分化 4。 2007年， C.E. Murry等利用单层培养的人类胚胎干细胞直接诱 导分化心肌细胞用于小鼠的心脏移植， 其平均的分化效率只有大约 30%, 即最后心肌细胞占总细胞数的 30%7。 经过密度梯度离心的分离 纯化，最终可以获得纯度 80%左右的心肌细胞群体。 2008年， GM. Keller 等利用悬浮培养类胚体进行分化， 在分化第六天用流式细胞术分离出 心肌的前体细胞继续培养分化， 大幅提高了分化效率， 最终分化效率 可以达到 50%以上 8。 但此法同样没能实现心房和心室肌细胞的定向分 化。 总之， 上述的分化心肌细胞方法中均尚未述及分化过程对心房细 胞和心室细胞发育的影响， 最终的分化产物大多报道是含有三类心肌 细胞的混合物， 而且目前没有分离这三类心肌细胞的特异分离方法的 相关报道。 2007年， 有报道利用慢病毒转染人类胚胎干细胞， 构建心 室细胞特异表达的 MLC-2v基因一段保守的启动子所启动的绿色荧光 蛋白（EGFP )插入的细胞系， 将其分化为表达绿色荧光蛋白的心肌细 胞。 该方法有利于心肌细胞的纯化， 纯度能达到 90%以上， 但由于该 类细胞需插入 MLC-2v诱导的 GFP或其他筛选基因的 DNA到干细胞的 基因组中， 因而这类具有转基因的心肌细胞并不适用于临床的细胞移 植。 2010年， 中国科学院生物物理研究所马跃实验室的研究发现， 在 干细胞心肌分化的中期， 即从中胚层细胞到心肌细胞的阶段（从干细 胞到中胚层细胞的阶段为分化初期；)，利用维甲酸处理分化的细胞可以 有效地将分化的干细胞诱导成心房肌细胞； 于此同时抑制维甲酸通路 则可以有效地将细胞分化成心室肌细胞 9。 虽然这一研究成果首次解释 了干细胞心肌分化是诱导获得心房肌细胞和心室肌细胞的方法， 但对 于如何诱导心室肌的分化尚未找出明确的主动调节分子。
发明内容
本发明的目的是提供体外诱导多能干细胞分化为心室肌细胞的 方法。
为了实现本发明目的，本发明的体外诱导多能干细胞分化为心室 肌细胞的方法是通过在体外维持、 扩增、 培养多能干细胞， 并于多能 干细胞分化的中期向培养基中加入可直接或间接激活 Smadl/5/8信号 通路的物质，使其定向分化为心室肌细胞。其中，所述激活 Smadl/5/8 信号通路是指使胞质中的 Smadl、 Smad5、 Smad8蛋白中的一种或几 种磷酸化。所述多能干细胞分化的中期即由中胚层细胞或心肌前体细 胞向心肌细胞分化的时期， 具体指从细胞开始表达 Brachyury (T)基因 和 /或 Mespl基因， 到分化成能自主收缩的心肌细胞之前的时期。
前述方法中， 所述的多能干细胞为胚胎干细胞 （embryonic stem cells )、 诱导的多能干细胞（induced pluripotent stem cells)、胚胎生殖细 胞或成人多能干细胞， 且这些多能干细胞来自于人类或哺乳动物。
前述方法中， 所述可直接或间接激活 Smadl/5/8信号通路的物质 和 /或骨
Figure imgf000005_0001
其终浓 度为 0.01-1200ng/mL。
前述方法中， 在多能干细胞分化的初期， 即从干细胞分化成心肌 前体细胞的时期， 向培养基中加入具有促进心肌细胞分化的物质。 所 述具有促进心肌细胞分化的物质为 BMP4、 碱性成纤维细胞生长因子 ( bFGF )、 激活素 A ( Activin A )、 Noggin, Dorsomorphin、 6-溴靛玉 红 -3'-肟 ( 6-bromoindirubin-3'-oxime, BIO ) 等中的至少一种。 其中， 添加的细胞生长因子的终浓度为 0.01-1200ng/mL, 其它小分子化合物 的终浓度为 0.001-100μΜ。
前述方法中， 在多能干细胞分化的中期， 还向培养基中添加具有 抑制 Wnt信号通路的物质。 所述具有抑制 Wnt信号通路的物质为 dickkopf同源物 1 ( dickkopf homolog 1 , DKKl ), IWP、 IWR ( inhibitors of Wntresponse ) 等中的至少一种， 添加的小分子化合物的终浓度为 0.001-100μΜ , DKKl的终浓度为 0.01 - 1200ng/mL。
前述方法中， 在多能干细胞分化的中期， 还包括 i ) 当培养基中 不含有维甲酸或其合成所需的底物时， 向其中加入维甲酸受体 γ ( RARy )的激活剂； 或 ii )当培养基中含有维甲酸或其合成所需的底 物时， 向其中加入维甲酸受体 a ( RARa )和 /或维甲酸受体 β ( RARJ3 ) 的拮抗剂。 i ) 中所述激活剂以及 ii ) 中所述拮抗剂的终浓度为 0.001-100μΜ。
具体地， 本发明提供三套技术方案：
技术方案 I：
(1)悬浮培养或单层培养实质上未分化的多能干细胞；
(2)在分化过程的初期加入具有促进心肌细胞分化的细胞因子，如 BMP4、 bFGF、 ActivinA, Noggin等； 或加入具有抑制 BMP通路的小 分子化合物， 如 Dorsomorphin等； 或加入能够激活 Wnt3a的小分子化 合物， 如 BIO、 CHIR99021等；
(3)在分化过程的中期通过向培养基中添加例如 DKK1、 IWP或 IWR等抑制 Wnt通路的蛋白生长因子或小分子化合物以抑制 Wnt信号 通路， 与此同时加入 BMP2和 /或 BMP4等能够激活 Smadl/5/8磷酸化的 信号分子， 定向诱导细胞分化成为心室肌细胞。
技术方案 II：
(1)悬浮培养或单层培养实质上未分化的多能干细胞；
(2)在分化过程的初期加入具有促进心肌细胞分化的细胞因子，如 BMP4、 bFGF、 ActivinA, Noggin等； 或加入具有抑制 BMP通路的小 分子化合物， 如 Dorsomorphin等； 或加入能够激活 Wnt3a的小分子化 合物， 如 BIO等；
(3)在分化过程的中期通过向培养基中添加例如 DKK1、 IWP或 IWR等抑制 Wnt通路的蛋白生长因子或小分子化合物以抑制 Wnt信号 通路， 与此同时加入能够刺激细胞表达分泌能激活 Smadl/5/8磷酸化 (1)悬浮培养或单层培养实质上未分化的多能干细胞；
(2)在分化过程的初期加入具有促进心肌细胞分化的细胞因子，如 BMP4、 bFGF、 Activin A, Noggin等； 或加入具有抑制 BMP通路的小 分子化合物， 如 Dorsomorphin等； 或加入能够激活 Wnt3a的小分子化 合物， 如 BIO等；
(3)在分化过程的中期通过向培养基中添加例如 DKK1、 IWP或 IWR等抑制 Wnt通路的蛋白生长因子或小分子化合物以抑制 Wnt信号 通路， 与此同时， 在含有维甲酸或其合成底物的培养基中加入维甲酸 受体 RARa和 /或 RARP的拮抗剂（如 RARa的拮抗剂 Ro41-5253和 RARJ3 的拮抗剂 LE135 )。 利用这些步骤可以将干细胞分化的心肌细胞定向 诱导成以心室肌为主的心肌细胞。
分化过程从第 14天以后， 每 3天更换一次无因子的培养基； 培养 60-90天后， 利用膜片钳技术检测心肌细胞的动作电位， 钙信号影像 记录， MLC-2v和 cTNT双染并进行流式细胞分析等方法确定分化的心 室肌细胞及非心室肌的心肌细胞 （包括心房肌细胞和起搏细胞）。
本发明进一步提供釆用上述方法制备的心室肌细胞在筛选治疗 心脏病药物及药物的心脏毒理检测中的应用。
本发明进一步提供釆用上述方法制备的心室肌细胞在受损心脏 的干细胞治疗中的应用。
本发明还提供了一套促进干细胞分化为心室肌细胞的方法，该方 法包括激活中胚层细胞中的 Smadl/5/8信号通路； 其中， 所述中胚层 细胞由干细胞分化而来。
前述方法中， 所述干细胞为全能干细胞 （ Totipotent stem cell )、 多能干细胞 ( Pluripotent stem cell )、 专能干细胞 ( Multipotent stem cell ), 寡能干细胞 （ Oligopotent stem cell )或单能干细胞 （ Unipotent stem cell )。
前述方法中， 所述干细胞为胚胎干细胞、 诱导的多能干细胞、 胎 儿干细胞或成体干细胞。
前述方法中， 所述干细胞为哺乳动物干细胞。
前述方法中， 所述哺乳动物干细胞为人类干细胞。
前述方法中， 所述干细胞为人胚胎干细胞或诱导的人多能干细胞。 前述方法中， 由干细胞分化成中胚层细胞是通过使未分化的干细 胞与以下物质中的至少一种发生接触； 所述物质为 bFGF、 骨形态发 生蛋白 2 ( BMP2 )、 BMP4、 Activin A、 BMP拮抗剂、 BMP信号通路 抑制剂、 Wnt3a信号通路激活剂等。
前述方法中， 所述 BMP拮抗剂为 BMP4拮抗剂。
前述方法中， 所述 BMP拮抗剂为 Noggin。
前述方法中， 所述 BMP信号通路抑制剂为小分子 BMP信号通路 抑制剂。
前述方法中， 所述小分子 BMP信号通路抑制剂为 Dorsomorphin。 前述方法中， 所述 Wnt3a信号通路激活剂为小分子 Wnt3a信号通 路激活剂。
前述方法中， 所述小分子 Wnt3a信号通路激活剂为 GSK-3a/p的 ATP-竟争性抑制剂。
前述方法中， 所述 GSK-301/β的 ATP-竟争性抑制剂为细胞渗透双 吲哚并 （靛玉红） 化合物 （ cell-permeable bw-indolo (indirubin) compound )。
前述方法中， 所述细胞渗透双吲哚并 （靛玉红） 化合物为 BIO。 前述方法中， 所述 bFGF、 BMP2、 BMP4、 Activin A、 BMP拮抗 剂、 BMP信号通路抑制剂、 Wnt3a信号通路激活剂等， 添加的生长因 子的终浓度为 0.01-1200ng/mL, 其它物质的终浓度为 0.001-100μΜ。
前述方法中， 使干细胞与 ΒΜΡ2和 /或 ΒΜΡ4接触从而激活 Smadl/5/8信号通路。
前述方法 中 ， BMP2和 I或 BMP4使用 的终浓度为 约 0.01-1200ng/mL。 前述方法中，通过在不含有维甲酸或其前体的培养基中培养干细 胞， 并使干细胞与维甲酸受体 γ ( RARy )激活剂发生接触， 从而激活 Smadl/5/8信号通路。
前述方法中， 所述维甲酸前体为维生素 A。
前述方法中， 所述 RARy激活剂为 BMS961、 Palovarotene或购自
SIGMA-ALDRICH的 CD437等。
前述方法中， RARy激活剂使用的终浓度为约 0.001-100μΜ。
前述方法中， 使干细胞与维甲酸受体 a ( RARa )和 /或维甲酸受 体 β ( RAR )拮抗剂发生接触， 从而激活 Smadl/5/8信号通路。
前述方法中， 所述 RARa拮抗剂为 Ro41-5253、 BMS 195614或 ER50891等， 所述 RARJ拮抗剂为 LE135等。
前述方法中， RARa和 I或 RARP拮抗剂使用的终浓度为 0.001-100μΜ。
前述方法中， 进一步包括使所述干细胞与 Wnt抑制剂发生接触， 从而使干细胞分化成心室肌细胞。
前述方法中， 所述 Wnt抑制剂为 DKK1、 IWP、 Wntresponse抑制 剂等中的至少一种。
前述方法中， Wnt抑制剂 DKK1使用的终浓度为 0.01-1200ng/mL, 其它抑制剂的终浓度为 0.001 - 100μΜ。
本发明还提供按照上述方法制备得到的心室肌细胞。
前述的心室肌细胞， 心室特异表达基因、 心室肌动作电位 ( embryonic ventricular-like action potentials , AP )和 /或心室肌细胞的 代表性特征 Ca2+火花 （Ca2+ spark ) 的表达水平或比率升高。
前述的心室肌细胞， 所述心室特异表达基因为 IRX-4和 /或 MLC-2v。
本发明还提供一种组合物， 其包括已分化成中胚层细胞的， 且用 能够激活干细胞中 Smadl/5/8信号通路的外源性物质处理的干细胞。
前述的组合物， 所述能够激活干细胞中 Smadl/5/8信号通路的外 源性物质为 BMP2和 /或 BMP4等。
前述的组合物， 所述能够激活干细胞中 Smadl/5/8信号通路的外 源性物质为 RARy激活剂。
前述的组合物， 所述能够激活干细胞中 Smadl/5/8信号通路的外 源性物质为 RARa和 /或 RARJ拮抗剂。
本发明还提供由干细胞生成心室肌细胞的方法， 该方法包括： 1 ) 使干细胞与 bFGF和 BMP4接触， 从而引发细胞分化； 2 ) 经 bFGF和 BMP4处理后的干细胞与 ActivinA接触， 从而形成中胚层细胞； 3 )将 已分化成中胚层细胞的干细胞与 Noggin接触，从而提高干细胞向心肌 细胞分化的效率； 4 )激活经 Noggin处理后的干细胞中的 Smadl/5/8信 号通路， 从而促进心室肌细胞的形成； 5 )将经 Noggin处理后的干细 胞与以下物质中的至少一种接触， 使干细胞分化成心室肌细胞； 所述 物质为 DKK1、 IWP、 IWR等中的至少一种。
前述方法中， 通过使所述干细胞与 BMP2和 /或 BMP4接触， 从而 激活 Smadl/5/8信号通路。
前述方法中， 通过在不含有维甲酸或其前体维生素 A的培养基中 培养干细胞， 并使干细胞与 RARy激活剂发生接触， 以提高培养细胞 中 BMP2/4的表达水平， 从而激活 Smadl/5/8信号通路。
前述方法中，通过使所述干细胞与 RARa和 /或 RARP拮抗剂接触， 从而激活 Smadl/5/8信号通路。
本发明还提供按照上述方法制备得到的心室肌细胞。
本发明还提供用于治疗心脏受损或心脏病的药物组合物，其包括 有效量的前述心室肌细胞以及药用载体或赋形剂。
本发明还提供治疗心脏受损或心脏病的方法，该方法包括对需要 进行此类治疗的个体施以有效量的上述药物组合物。
前述方法中， 所述个体为人类。
前述方法用于心脏受损的干细胞治疗。
本发明还提供前述心室肌细胞在制备用于治疗和 /或预防心脏受 损或心脏病的药物中的应用。
本发明还提供前述心室肌细胞在筛选和 /或开发用于治疗和 /或预 防心脏受损或心脏病的药物中的应用。
本发明还提供前述心室肌细胞在药物的心脏毒理检测中的应用。 本发明进一步提供鉴定心室肌细胞调节因子的方法， 该方法包 括： 1 )使前述心室肌细胞与候选的调节因子接触， 并测定该候选调 节因子对心室肌细胞功能的影响； 2 ) 测定未与该候选调节因子接触 的心室肌细胞的功能；如果与该候选调节因子接触的心室肌细胞的功 能较未与该候选调节因子接触的心室肌细胞的功能不同，则确定该候 选调节因子为所述心室肌细胞的功能调节因子。
本发明提供的体外诱导多能干细胞分化为心室肌细胞的方法，通 过对干细胞的直接诱导， 在干细胞心肌分化的中期， 通过向培养体系 中直接加入 BMP2和 /或 BMP4等能够激活 Smadl/5/8信号通路的物质， 可以使干细胞定向分化成以心室肌细胞为主的心肌细胞。在培养体系 中， 在含有维甲酸或其合成所需的前体（如维生素 A ) 的条件下， 添 加 BMP2和或 BMP4等能够激活 Smad 1/5/8信号通路的物质可以有效地 抑制心肌前体细胞向心房肌细胞的分化， 并诱导心室肌细胞的分化。 在加入维甲酸或维生素 A的同时加入 BMP2和 /或 BMP4等能够激活 Smadl/5/8信号通路的物质， 则分化的心肌细胞中心房肌细胞的比率 随着加入 BMP4浓度的增加而降低， 而分化的心肌细胞中心室肌细胞 的比率却随之增加。 如果在干细胞心肌分化的中期， 只加入 BMP2/4, 同时在培养基中去除维甲酸或其合成所需的前体，则可以使心肌前体 细胞高效定向分化成心室肌细胞。 在干细胞分化的中期， 利用在培养 基中添加维甲酸的受体 RARa或 RARP的激活剂，可以有效地抑制 ι 室 肌细胞的分化， 表现为心室肌细胞的早期标记基因 IRX-4的表达受到 抑制。 但是在无维甲酸或其前体维生素 A的条件下， 加入 RARy的激 活剂可以有效地将干细胞诱导分化成心室肌细胞。 另外， 在含有维生 素 A或维甲酸的培养基中添加 RARa和 RARJ的拮抗剂也可以有效地 提升 IRX-4的表达水平， 诱导干细胞分化成心室肌细胞。
在本发明中， 明确阐明了干细胞心肌分化时， BMP、 Smadl/5/8 通路正向地调节心室肌细胞的分化，为后续利用干细胞分化获取高纯 度的心室肌细胞奠定了基础。
本发明的优点在于：
利用本发明提供的体外诱导多能干细胞分化为心室肌细胞的方 法， 无需通过任何纯化步骤， 即可成功获得高纯度的、 具有生物学活 性及功能的心室肌细胞，不仅为揭示心肌前体细胞向心室肌细胞分化 过程中的调节机制奠定了坚实的理论基础，而且对于细胞移植治疗心 肌梗死和利用人干细胞分化的心肌细胞进行药物研发具有重要意义。 附图说明
图 1为在干细胞分化中期， BMP信号通路的表达及对心室肌细胞 特异标记基因 IRX-4表达的作用； 其中， A为在干细胞心肌分化的第 5 和第 6天时利用 RT-PCR技术检测 BMP2、 BMP4及其受体的表达； B为 Westen blot 检测 BMP通路的下游信号 Smadl/5/8被磷酸化激活的情 况， P-Smadl/5/8为磷酸化的 Smadl/5/8蛋白分子， T- Smadl/5/8为总的 Smadl/5/8蛋白分子， β-肌动蛋白为内参蛋白； C中柱状图表示在分化 的第 14天定量 RT-PCR检测 IRX-4基因表达水平的实验结果，该结果显 示出在不同的时间段内向培养基中加入 ΙμΜ的维甲酸和 200ng/mL的 BMP4时 IRX-4的表达水平，连线标记显示相应条件下干细胞心肌分化 的效率； 其中， N表示 Noggin, B表示 BMP4, NVa为不含有维生素 A 的培养基处理的分化细胞， RA表示维甲酸， 数字表示浓度， BMP4 的浓度单位为 ng/mL, 定量 PCR的结果是与 GADPH表达量比较的相 对值。
图 2为定量 PCR检测分化的第 14天心室肌细胞早期特异表达基因 IRX-4在不同处理的分化干细胞中的表达水平； 其中， A显示在培养 基中加入不同浓度的 BMP4后， IRX-4的表达随着 BMP的浓度增加而 升高， 但在加入 BMP的拮抗剂 Noggin后表达水平下降； B为在不含维 甲酸前体维生素 A的培养基中加入不同剂量的 Noggin可以有效地降 低 IRX-4的表达水平； C为在 ΙμΜ维甲酸存在时， IRX-4的表达水平随 着加入 ΒΜΡ4的浓度的升高而增力口； D显示 ΒΜΡ4在维甲酸存在时提高 IRX-4表达的现象随着向培养体系中加入 Noggin的浓度的增加而降 低； 其中， N表示 Noggin, B表示 BMP4, NVa为无维生素 A培养基处 理的分化细胞， RA表示维甲酸， 数字表示浓度， 单位为 ng/mL。 定量 PCR的结果是与 GADPH表达量比较的相对值。
图 3为定量 RT-PCR分析 IRX-4基因在分化的第 14天的表达水平， 结果显示 BMP家族的其他成员在干细胞心肌分化的第 5-8天处理干细 胞同样可以有效地拮抗维甲酸对心室肌细胞早期表达基因 IRX-4的抑 制作用， 而且拮抗作用随加入 BMP家族生长因子的剂量的增加而加 大。 RA表示维甲酸， 数字表示生长因子的浓度， 单位为 ng/mL, 维甲 酸的浓度为 1μΜ。 定量 PCR的结果是与 GADPH表达量比较的相对值。
图 4显示经过维甲酸、 ΒΜΡ4和 Noggin处理的干细胞分化为成熟的 心室肌细胞时特异表达基因 MLC-2v的表达； 其中， A为利用 Westen blot检测经维甲酸、 BMP4诱导的干细胞分化的心肌细胞在分化 90天后 MLC-2v的表达； B为经维甲酸、 BMP4和 Noggin分别处理的分化 90天 后心肌细胞 cTNT和 MLC-2v双染的免疫荧光染色结果； 其中， B表示 BMP4, NVa为无维生素 A培养基处理的分化细胞， RA表示 ΙμΜ维甲 酸， 数字表示浓度， 单位为 ng/mL。
图 5为对分化的心肌细胞的钙活动进行共聚焦图像扫描和动作电 位联用记录并利用各类心肌细胞特异钙活模式对分化的心肌细胞分 类的结果； A显示具有心室肌动作电位 (ventricular-like action potential) 的心肌细胞具有钙火花 (Ca2+ Spark), 具有心房肌动作电位 (atrial-like action potential)的心肌细胞具有钙瞬变 (Ca2+Transient)的特征， 而具有 起搏细胞动作电位 (Nodal-like action potential)的心肌细胞具有钙震荡 (Ca2+ Oscillation)的特征； B为利用 A中得到的不同亚型心肌细胞的钙 信号模式，对不同处理组得到的心肌细胞进行分类所得到的各组中心 肌细胞具有钙火花、 钙瞬变和钙震荡的比率， 纵坐标为检测到的心肌 细胞中具有三种不同类型钙活动所占比率； RA表示 ΙμΜ的维甲酸， Β 表示 ΒΜΡ4, NVa表示无维生素 Α培养基， N表示 Noggin, 数字表示浓 度， 单位为 ng/mL。
图 6为定量 RT-PCR检测在细胞分化第 6天时在经 RARy激活剂处 理的细胞中 BMP2的表达水平。其中，定量 PCR的结果是与 GADPH表 达量比较的相对值。 NVa表示无维生素 A培养基。
图 7为在干细胞心肌分化中期， 向不含有维生素 A的培养基中加 入不同的维甲酸受体的激活剂或抑制剂等调节剂后， 在分化的第 14 天定量 RT-PCR分析心室肌特异基因 IRX-4在分化 14天的表达水平。其 中， RA表示维甲酸， RAi表示维甲酸的抑制剂 BMS189453 , NVa表示 培养基中不含有维生素八。 RAR-pan拮抗剂为 BMS493。定量 PCR的结 果是与 cTNT表达量比较的相对值。
图 8为对经不同诱导分化的心肌细胞的单细胞动作点位和成熟心 室肌细胞特异标记基因 MLC-2v的鉴定。 A为经不同诱导条件分化的 心肌细胞中具有心房肌、 心室肌和起搏细胞动作电位的细胞的比率 ( n>30 )。 B为流式细胞分析仪检测经不同条件处理的 90天分化细胞 群体内心肌细胞 （cTNT阳性细胞） 中表达 MLC-2v细胞的比率。 RA 表示 ΙμΜ的维甲酸， Β表示 ΒΜΡ4, NVa表示无维生素 Α培养基， N表 示 Noggin, 数字表示浓度， 单位为 ng/mL。 RARy的浓度为 0.1μΜ。
图 9为本发明实施例 2的体外诱导多能干细胞分化为心室肌细胞 的过程。
图 10为本发明实施例 3中体外诱导多能干细胞分化为心室肌细胞 的过程。
图 11为本发明实施例 4中体外诱导多能干细胞分化为心室肌细胞 的过程。
图 12为经维甲酸处理后分化的心肌细胞， 作 cTnT和 MLC-2v免疫 荧光共染色的结果。
图 13为经维甲酸和 200ng/mL BMP4处理后分化的心肌细胞， 作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 14为经无维生素 A的培养基及 1200ng/mL Noggin处理后分化的 心肌细胞， 作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 15为实施例 2中最终获得的分化的心肌细胞，作 cTnT和 MLC-2v 免疫荧光共染色的结果。
图 16为经无维生素 A的培养基处理后分化的心肌细胞，作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 17为实施例 3中最终获得的分化的心肌细胞，作 cTnT和 MLC-2v 免疫荧光共染色的结果。
图 12-图 17中， 带" *"标记的细胞为非心室肌心肌细胞， 带" Λ "标记 的细胞为表达 MLC-2v的心室肌细胞。 具体实施方式
以下实施例用于说明本发明， 但不用来限制本发明的范围。 若未 特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规 手段， 所用原料均为市售商品。
以下实施例中使用的人胚胎干细胞系 H7购自 WiCell Research Institute,美国； B27培养基添加物和 RPMI1640培养基，购自 Invitrogen; Activin A, bFGF、 DKK1、 BMP4及 Noggin均购自 R&D systems。
实施例 1 BMP/Smadl/5/8信号通路在诱导心肌前体细胞向心室肌 细胞分化中的作用
1 研究发现， 在干细胞心肌分化的过程中， 在决定心肌细胞亚型 分化时期， 向培养基中加入维甲酸或其合成所需的底物如维生素 A, 可以将干细胞定向诱导成为心房肌细胞；若在此时向培养基中加入维 甲酸的抑制剂或在培养基中去除维甲酸的合成底物维生素 A, 可以定 向地将干细胞分化成心室肌细胞 9。 通过 RT-PCR反应， 分析分化的人 胚胎干细胞在分化的中期表达 BMP2/4及其相应的受体， 结果如图 1所 示， BMP通路的配体和受体都存在于培养的细胞中。利用 Western blot 检测 BMP2/4下游的信号分子磷酸化 Smadl/5/8, 实验发现在添加维甲 酸以及无维甲酸或无维生素 A的培养条件下， 分化细胞的 Smadl/5/8 都被磷酸化 （图 1 ), 证明 BMP通路在干细胞心肌分化的中期被激活。 这些实验结果显示在干细胞分化的第 5至第 8天 BMP通路有可能参与 调节新机前体细胞向心室肌细胞的分化。
2 进一步检测 BMP通路在干细胞心肌分化中心肌亚型定向分化 的作用。已知 IRX-4是心室肌细胞分化过程中早期表达的标志性基因。 因此本发明通过检测 IRX-4的表达水平来进一步研究 BMP信号通路在 心肌亚型决定时的作用。
实验发现， 在干细胞分化的中期在无维生素 A培养基分化的干细 胞中加入 BMP2/4通路的抑制剂 Noggin , 可以有效地降低分化细胞中 IRX-4的表达， 并且 IRX-4的表达水平随着 Noggin的用量加大 ( 300ng/mL、 600ng/mL和 1200ng/mL ) 而降低， 如图 2所示。 3 实验表明，在干细胞心肌分化的中期加入维甲酸可以抑制心室 肌特异表达基因 IRX-4的表达。 在干细胞心肌分化的中期， 在加入维 甲酸的同时加入不同剂量的 BMP4, 然后在第 14天时利用定量 PCR检 测 IRX-4的表达。 结果（图 2 )表明， 在经维甲酸处理的干细胞心肌分 化中加入 BMP可以提高 IRX-4的表达， 且随着添加 BMP4量的提高， IRX-4的表达水平亦随之增加。
BMP家族的其他成员也能够拮抗维甲酸对 IRX-4的抑制作用。 BMP家族的成员多具有相似的功能， 如图 3所示， 定量 RT-PCR实验证 明， 在干细胞心肌分化的中期， 在有 ΙμΜ维甲酸的情况下， BMP家族 的其他成员也能够不同程度地提高 IRX-4的表达水平，而且 IRX-4的表 达水平随着加入生长因子的浓度的升高而升高。
综上所述， 通过对心室肌细胞早期特异性表达基因 IRX-4的研究 发现， BMP信号通路可以有效地提高分化的心肌细胞中 IRX-4的表达， 从而揭示 BMP信号通路参与了干细胞分化过程中心室肌细胞早期分 化， 促进心室肌细胞的分化。
4 为了进一步验证 BMP通路调节心室肌细胞的分化功能，本发明 对分化 60-90天的心肌细胞的钙活动进行了共聚焦图像扫描研究。 心 室肌细胞与心房肌细胞和起搏细胞的钙活动有着明显的区别。心室肌 细胞的钙活动的成像频率较快， 被称之为"钙火花（Ca+ sparks ) "； 而 心房肌细胞的钙活动的成像显示其频率较慢， 且信号较大， 被称之为 "钙瞬变 （Ca+ transients ) "； 起搏细胞钙活动的成像显示其具有明显 的周期性， 被称之为"钙震荡"。 首先通过单细胞膜片钳与共聚焦图像 扫描连用，发现在膜片钳记录的具有心室肌动作电位的 20个细胞的钙 活动全部为钙火花；在膜片钳记录的具有心房肌动作电位的 20个细胞 全部具有钙瞬变；在膜片钳记录的具有起搏细胞动作电位的 20个细胞 全部具有钙震荡。 因此， 比较心肌细胞的钙活动的成像可以有效地区 分心室肌细胞与心房肌细胞和起搏细胞。 钙成像结果显示， 经维甲酸 处理的分化的心肌细胞中多数具有钙瞬变，且钙瞬变的比率随着加入 BMP4浓度的增加而降低， 相反地， 分化的心肌细胞中具有钙火花的 细胞比率随着添加 BMP4浓度的增加而增加。 该结果表明， 激活 BMP 信号通路可以有效地诱导干细胞分化成心室肌细胞 (图 5 )。
5 如前所述， 在心肌细胞分化的早期， IRX-4是心室肌细胞分化 的重要的特异表达基因。 随着心肌细胞的成熟， 心室肌细胞开始特异 性地表达 MLC-2v (肌球蛋白轻链 2 )基因。 因此， 本发明还检测了经 不同生长因子处理的培养 90天的分化细胞中 MLC-2v的表达水平。 Western blot显示， 在分化 90天的细胞中 MLC-2v的蛋白表达水平也随 着添加 BMP4的浓度的增加而增加 （图 4 ); 本发明还利用流式细胞仪 检测了 90天分化的细胞中表达 MLC-2v的心室肌细胞占所分化的心肌 细胞（表达 cTNT的细胞）的比率。 从图 8的结果可以看出， 在经维甲 酸处理的分化的细胞中加入 BMP处理可以有效地提高心肌细胞中表 达 MLC-2v细胞的比率， 并且在单独用 BMP4处理的条件下， 表达 MLC-2v细胞的比率达到最高。 鉴别心肌细胞亚型最经典的方法是测 量心肌细胞的动作点位。 通过检测经过维甲酸和不同剂量的 BMP4处 理的分化的心肌细胞中具有心房、心室肌细胞和起搏细胞动作电位的 比率发现（图 8 ), 在经维甲酸处理的分化细胞中， 随着添加 BMP4剂 量的增加， 具有心室肌动作点位的细胞的比率显著增加。 在单独加入 BMP4的分化细胞中 90%以上的心肌细胞具有心室肌动作电位， 即 90%以上的心肌细胞为心室肌细胞。
6研究表明，在胚胎干细胞心肌分化的早期加入 BMP2/4、 Activin A、 bFGF和 /或 Noggin, 并在分化的中期加入 DKK1等生长因子可以将 干细胞高效诱导分化成心肌细胞。 定量 RT-PCR实验结果显示在干细 胞心肌分化的中期， 在加入 DKK1的同时， 在不含维生素 A的 B27/RPMI1640培养基中， 如果加入维甲酸受体 RARa和 RARJ的激活 剂， 可以降低心室特异表达基因 IRX-4的表达水平（图 7 ); 但是在加 入 DKK1的同时加入 RARy的激活剂， 则可以诱导 BMP2和 IRX-4的高 表达（图 6和 7 ); 另外在培养基中有维生素 A的条件下， 在加入 DKK1 时同时抑制维甲酸受体 RARa和 RARP,则可以激活心室肌早期特异表 达基因 IRX-4的表达， 显示将干细胞诱导成为心室肌细胞。 由于维甲 酸共有三个 RAR受体， RARou RARp和 RARy, 在有维生素 A或维甲 酸的情况下， 同时抑制 RARa和 RARP, 与单独激活 RARy有相似的机 制与效果。
流式细胞仪检测实验证明 （图 8 ), 在经 RARy诱导的 90天分化的 心肌细胞中表达 MLC-2v的心室肌细胞占所分化的心肌细胞 （表达 cTNT的细胞） 的比率可以达到 80%以上。 另外对经 RARy诱导的 90天 分化的心肌细胞进行电生理动作电位的鉴别， 显示分化细胞中 92%的 心肌细胞具有心室肌动作电位， 是心室肌细胞。
实施例 2 体外诱导多能干细胞分化为心室肌细胞（技术方案一） 将人胚胎干细胞 H7平铺于含明胶（Gelatin )的培养皿中， 加入含 B27(l X浓度）的 RPMI1640培养基在 37 °C C02细胞培养箱中培养。 心 肌分化的过程如图 9所示，从第 0天到第 3天，向培养基中加入 Activin A ( 10ng/mL ) ,BMP4(6ng/mL)和 bFGF(6ng/mL)。 在第 3天结束时更换培 养基， 同时加入 BMP2/4的抑制剂 Noggin ( 3 OOng/mL )。 在第 5天结束 时， 将培养基更换为不添加维生素 A的含 B27的 RPMI1640培养基。 同 时在培养基中加入 Wnt3a的抑制剂 DKK1 ( 300ng/mL ) 和 BMP4(10ng/mL)。 在分化的第 8天结束时， 将培养基更换为只含有 300ng/mL DKKl的培养基， 在分化第 10天结束时更换培养基， 不添加 任何生长因子。 以后每 3天更换一次添加 B27的 RPMI1640培养基。 14 天时可以观察到大量跳动的心肌细胞。 方案流程见图 9。
实施例 3 体外诱导多能干细胞分化为心室肌细胞（技术方案二） 将人胚胎干细胞 H7平铺于含明胶（Gelatin )的培养皿中， 加入含 B27 ( 1 X浓度 ) 的 RPMI1640培养基在 37°C C02细胞培养箱中培养。 心肌分化的过程如图 10所示， 从第 0天到第 3天， 向培养基中加入 Activin A ( 10ng/mL ), BMP4(6ng/mL)和 bFGF(6ng/mL)。 在第 3天结 束时， 更换培养基， 同时加入 BMP2/4的抑制剂 Noggin ( 300ng/mL )。 在第 5天结束时， 将培养基更换为不添加维生素 A的含 B27的 RPMI1640培养基。 同时在培养基中加入 Wnt3a的抑制剂 DKK1 ( 300ng/mL ) 和维甲酸受体 RARy的激活剂 BMS961 ( Ο. Ι μΜ , 购自 Tocris )。 在分化的第 8天结束时， 将培养基更换为只含有 300ng/mL DKKl的培养基， 在分化第 10天结束时更换培养基， 不添加任何生长 因子。 以后每 3天更换一次添加 B27的 RPMI1640培养基。 14天时可以 观察到大量的跳动心肌细胞。 方案流程见图 10。
实施例 4 体外诱导多能干细胞分化为心室肌细胞（技术方案三） 将人胚胎干细胞 H7平铺于含明胶（Gelatin ) 的培养皿中， 加入含 B27 ( 1 X浓度 ) 的 RPMI1640培养基在 37°C C02细胞培养箱中培养。 心肌分化的过程如图 11所示， 从第 0天到第 3天， 向培养基中加入 Activin A ( 10ng/mL ), BMP4(6ng/mL)和 bFGF(6ng/mL)。 在第 3天结 束时， 更换培养基， 同时加入 BMP2/4的抑制剂 Noggin ( 300ng/mL )。 在第 5天结束时， 使用含有维生素 A的含 B27的 RPMI1640培养基。 同 时在培养基中加入 Wnt3a的抑制剂 DKKl ( 300ng/mL ) 和维甲酸受体 RARa和 β的拮抗剂 BMS 195614和 LE135 (浓度分别为 0.1 μΜ和 0.5μΜ )。 在分化的第 8天结束时， 将培养基更换为只含有 300ng/mL DKKl的培养基， 在分化第 10天结束时更换培养基， 不添加任何生长 因子。 以后每 3天更换一次添加 B27的 RPMI1640培养基。 14天时可以 观察到大量跳动的心肌细胞。 方案流程见图 11。
图 12为经维甲酸处理后分化的心肌细胞， 作 cTnT和 MLC-2v免疫 荧光共染色的结果。
图 13为经维甲酸和 200ng/mL BMP4处理后分化的心肌细胞， 作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 14为经无维生素 A的培养基及 1200ng/mL Noggin处理后分化的 心肌细胞， 作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 15为实施例 2中最终获得的分化的心肌细胞，作 cTnT和 MLC-2v 免疫荧光共染色的结果。
图 16为经无维生素 A的培养基处理后分化的心肌细胞，作 cTnT和 MLC-2v免疫荧光共染色的结果。
图 17为实施例 3中最终获得的分化的心肌细胞，作 cTnT和 MLC-2v 免疫荧光共染色的结果。
以上图 12-图 17中， 带" *"标记的细胞为非心室肌心肌细胞， 带" Λ" 标记的细胞为表达 MLC-2v的心室肌细胞。
对实施例 2和 3中分化 60-90天的心肌细胞的钙活动进行了共聚焦 图像扫描研究， 钙成像结果如图 5所示， 从图 5可以直接计算得出分化 的心肌细胞中具有钙火花的细胞占分化细胞总数的比率。
以上实施例中涉及到的培养基中各添加物的终浓度，其有效范围 为生长因子的终浓度在 0.01-1200ng/mL , 小分子化合物的浓度在 0.001-100μΜ。
利用本发明方法，成功获得了具有生物学活性及功能的心室肌细 胞， 不仅揭示了心肌前体细胞向心室肌细胞分化过程中的调节机制， 而且分化得到的人心室肌细胞对细胞移植治疗心肌梗死和心脏毒理 分析及心脏相关药物的研发有广泛的应用。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详 尽的描述， 但在本发明基础上， 可以对之作一些修改或改进， 这对本 领域技术人员而言是显而易见的。 因此， 在不偏离本发明精神的基础 上所做的这些修改或改进， 均属于本发明要求保护的范围。 工业实用性 釆用本发明提供的体外诱导多能干细胞分化为心室肌细胞的方 法， 成功获得了具有生物学活性及功能的心室肌细胞， 不仅揭示了心 肌前体细胞向心室肌细胞分化过程中的调节机制，而且分化得到的人 心室肌细胞对细胞移植治疗心肌梗死和心脏毒理分析及心脏相关药 物的研发有广泛的应用。
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Claims

权 利 要 求 书
1. 体外诱导多能干细胞分化为心室肌细胞的方法， 其是通过在 体外维持、 扩增、 培养多能干细胞， 使其定向分化为心室肌细胞， 其 特征在于，在多能干细胞分化的中期即由中胚层细胞或心肌前体细胞 向心肌细胞分化的时期， 向培养基中加入可直接或间接激活 Smadl/5/8信号通路的物质。
2. 根据权利要求 1所述的方法， 其特征在于， 所述多能干细胞为 来自于人类或哺乳动物的胚胎干细胞或诱导的多能干细胞。
3. 根据权利要求 1所述的方法， 其特征在于， 所述可直接或间接 激活 Smadl/5/8信号通路的物质为骨形态发生蛋白 2 ( BMP2 )和 /或骨 形态发生蛋白 4 ( BMP4 ), 其终浓度为 0.01-1200ng/mL。
4. 根据权利要求 1-3任一所述的方法， 其特征在于， 在多能干细 胞分化的初期，向培养基中加入具有促进心肌细胞分化的物质；其中， 多能干细胞分化的初期是指从干细胞分化成中胚层细胞的时期。
5. 根据权利要求 4所述的方法， 其特征在于， 所述具有促进心肌 细胞分化的物质为 BMP4、 碱性成纤维细胞生长因子 （bFGF )、 激活
^ A ( Activin A )、 Noggin、 Dorsomorphin、 6-溴靛玉红 -3'-月亏 ( 6-bromoindirubin-3 '-oxime , BIO ) 中的至少一种；
其中， 添加的生长因子的终浓度为 0.01-1200ng/mL, 其它物质的 终浓度为 0.001-100μΜ。
6. 根据权利要求 1-3任一所述的方法， 其特征在于， 在多能干细 胞分化的中期， 还向培养基中添加具有抑制 Wnt信号通路的物质。
7. 根据权利要求 6所述的方法， 其特征在于， 所述具有抑制 Wnt 信号通路的物质为 dickkopf同源物 1 ( dickkopf homolog 1 , DKK1 )、 IWP、 Wntresponse的抑制剂 （IWR ) 中的至少一种； 其中， 添加的 DKK1 的终浓度为 0.01-1200ng/mL , 其它物质的终浓度为 0.001-100μΜ。
8. 根据权利要求 6所述的方法， 其特征在于， 在多能干细胞分化 的中期， 还包括 i)当培养基中不含有维甲酸或其合成所需的底物时， 向其中加入维甲酸受体 γ ( RARy ) 的激活剂； 或 ii)当培养基中含有维 甲酸或其合成所需的底物时， 向其中加入维甲酸受体 a ( RARa ) 和 / 或维甲酸受体 β ( RAR ) 的拮抗剂；
其中， i)中所述激活剂和 ii)中所述拮抗剂的终浓度范围为 0.001-100μΜ。
9. 根据权利要求 1-8任一项所述方法制备的心室肌细胞在筛选治 疗心脏病药物中的应用。
10. 根据权利要求 1-8任一项所述的方法制备的心室肌细胞在药 物的心脏毒理检测中的应用。
11. 根据权利要求 1-8任一项所述的方法制备的心室肌细胞在受 损心脏的干细胞治疗中的应用。
12. 促进干细胞分化为心室肌细胞的方法， 该方法包括激活中胚 层细胞中的 Smadl/5/8信号通路； 其中， 所述中胚层细胞由干细胞分 化而来。
13. 根据权利要求 12所述的方法， 其特征在于， 所述干细胞为全 能干细胞 ( Totipotent stem cell ), 多能干细胞 ( Pluripotent stem cell ), 专能干细胞（ Multipotent stem cell )、寡能干细胞（ Oligopotent stem cell ) 或单能干细胞 （ Unipotent stem cell )。
14. 根据权利要求 12所述的方法， 其特征在于， 所述干细胞为胚 胎干细胞、 诱导的多能干细胞、 胎儿干细胞或成体干细胞。
15. 根据权利要求 12所述的方法， 其特征在于， 所述干细胞为哺 乳动物干细胞。
16. 根据权利要求 15所述的方法， 其特征在于， 所述哺乳动物干 细胞为人类干细胞。
17. 根据权利要求 12所述的方法， 其特征在于， 所述干细胞为人 胚胎干细胞或诱导的人多能干细胞。
18. 根据权利要求 12-17任一项所述的方法， 其特征在于， 由干 细胞分化成中胚层细胞是通过使未分化的干细胞与以下物质中的至 少一种发生接触； 所述物质为 bFGF、 BMP2、 BMP4、 ActivinA、 BMP 拮抗剂、 BMP信号通路抑制剂、 Wnt3a信号通路激活剂。
19. 根据权利要求 18所述的方法， 其特征在于， 所述 BMP拮抗剂 为 BMP4拮抗剂。
20. 根据权利要求 19所述的方法， 其特征在于， 所述 BMP拮抗剂 为 Noggin。
21. 根据权利要求 18所述的方法， 其特征在于， 所述 BMP信号通 路抑制剂为小分子 BMP信号通路抑制剂。
22. 根据权利要求 21所述的方法， 其特征在于， 所述小分子 BMP 信号通路抑制刻为 Dorsomorphin。
23. 根据权利要求 18所述的方法， 其特征在于， 所述 Wnt3a信号 通路激活剂为小分子 Wnt3a信号通路激活剂。
24.根据权利要求 23所述的方法，其特征在于，所述小分子 Wnt3a 信号通路激活剂为 GSK-3a/p的 ATP-竟争性抑制剂。
25. 根据权利要求 24所述的方法， 其特征在于， 所述 GSK-3a/p 的 ATP-竟争性抑制剂为细胞渗透双吲哚并 （靛玉红） 化合物。
26. 根据权利要求 25所述的方法， 其特征在于， 所述细胞渗透双 吲哚并 (靛玉红) 化合物为 BIO。
27. 根据权利要求 18-26任一项所述的方法， 其特征在于， 添加 的生长因子的终浓度为 0.01-1200ng/mL , 其它物质的终浓度为 0.001-100μΜ。
28. 根据权利要求 12-27任一项所述的方法， 其特征在于， 使干 细胞与 ΒΜΡ2和 /或 ΒΜΡ4接触从而激活 Smadl/5/8信号通路。
29.根据权利要求 28所述的方法，其特征在于， BMP2和 /或 BMP4 使用的终浓度为 0.01-1200ng/mL。
30. 根据权利要求 12-27任一项所述的方法， 其特征在于， 通过 在不含有维甲酸或其前体的培养基中培养干细胞， 并使干细胞与 RARy激活剂发生接触， 从而激活 Smadl/5/8信号通路。
31. 根据权利要求 30所述的方法， 其特征在于， 所述维甲酸前体 为维生素八。
32. 根据权利要求 30或 31所述的方法， 其特征在于， 所述 RARy 激活剂为 BMS961、 Palovarotene或购自 SIGMA-ALDRICH的 CD437。
33. 根据权利要求 30-32任一项所述的方法， 其特征在于， RARy 激活剂使用的终浓度为 0.001-100μΜ。
34. 根据权利要求 12-27任一项所述的方法， 其特征在于， 使干 细胞与 RARa和 /或 RARJ拮抗剂发生接触， 从而激活 Smadl/5/8信号通
35. 根据权利要求 34所述的方法， 其特征在于， 所述 RARa拮抗 剂为 Ro41-5253、 BMS195614或 ER50891 ,所述 RARJ拮抗剂为 LE135。
36. 根据权利要求 33-35任一项所述的方法， 其特征在于， RARa 和 /或 RARJ拮抗剂使用的终浓度为 0.001-100μΜ。
37. 根据权利要求 12-36任一项所述的方法， 其特征在于， 进一 步包括使所述干细胞与 Wnt抑制剂发生接触， 从而使干细胞分化成心 室肌细胞。
38. 根据权利要求 37所述的方法， 其特征在于， 所述 Wnt抑制剂 为 DKK1、 IWP、 Wntresponse抑制剂中的至少一种。
39. 根据权利要求 37或 38所述的方法， 其特征在于， Wnt抑制剂 DKK1使用的终浓度为 0.01-1200ng/mL , 其它物质的终浓度为 0.001-100μΜ。
40. 按照权利要求 12-39任一项所述方法制备得到的心室肌细胞。
41. 根据权利要求 40所述的心室肌细胞， 其特征在于， 心室特异 表达基因、心室肌动作电位和 /或心室肌细胞的代表性特征 Ca2+火花的 表达水平或所占心肌细胞的比率升高。
42. 根据权利要求 41所述的心室肌细胞， 其特征在于， 所述心室 特异表达基因为 IRX-4和 /或 MLC-2v。
43. —种组合物， 其包括已分化成中胚层细胞的， 且用能够激活 干细胞中 Smadl/5/8信号通路的外源性物质处理的干细胞。
44. 根据权利要求 43所述的组合物， 其特征在于， 所述能够激活 干细胞中 Smadl/5/8信号通路的外源性物质为 BMP2和 /或 BMP4。
45. 根据权利要求 43所述的组合物， 其特征在于， 所述能够激活 干细胞中 Smadl/5/8信号通路的外源性物质为 RARy激活剂。
46. 根据权利要求 43所述的组合物， 其特征在于， 所述能够激活干 细胞中 Smadl/5/8信号通路的外源性物质为 RARa和 /或 RARP拮抗剂。
47. 由干细胞生成心室肌细胞的方法， 该方法包括：
1 )使干细胞与 bFGF和 BMP4接触， 从而引发细胞分化；
2 ) 经 bFGF和 BMP4处理后的干细胞与 Activin A接触， 从而形成 中胚层细胞；
3 )将已分化成中胚层细胞的干细胞与 Noggin接触， 从而提高干 细胞向心肌细胞分化的效率；
4 )激活经 Noggin处理后的干细胞中的 Smadl/5/8信号通路， 从而 促进心室肌细胞的形成；
5 )将经 Noggin处理后的干细胞与以下物质中的至少一种接触， 使干细胞分化成心室肌细胞； 所述物质为 DKK1、 IWP、 IWR中的至
48. 根据权利要求 47所述的方法， 其特征在于， 通过使所述干细 胞与 BMP2和 /或 BMP4接触， 从而激活 Smadl/5/8信号通路。
49. 根据权利要求 47所述的方法， 其特征在于， 通过在不含有维 甲酸或其前体的培养基中培养干细胞， 并使干细胞与 RARy激活剂发 生接触， 从而激活 Smadl/5/8信号通路。
50. 根据权利要求 47所述的方法， 其特征在于， 通过使所述干细 胞与 RARa和 /或 RARP拮抗剂接触， 从而激活 Smadl/5/8信号通路。
51. 按照权利要求 47-50任一项所述方法制备得到的心室肌细胞。
52. 用于治疗心脏受损或心脏病的药物组合物， 其包括有效量的 权利要求 40或 51所述的心室肌细胞以及药用载体或赋形剂。
53. 一种治疗心脏受损或心脏病的方法， 该方法包括对需要进行 此类治疗的个体施以有效量的权利要求 52所述的药物组合物。
54. 根据权利要求 53所述的方法，其特征在于，所述个体为人类。
55. 根据权利要求 53或 54所述的方法， 其特征在于， 该方法用于 心脏受损的干细胞治疗。
56. 权利要求 40或 51所述的心室肌细胞在制备用于治疗和 /或预 防心脏受损或心脏病的药物中的应用。
57. 权利要求 40或 51所述的心室肌细胞在筛选和 /或开发用于治 疗和 /或预防心脏受损或心脏病的药物中的应用。
58.权利要求 40或 51所述的心室肌细胞在药物的心脏毒理检测中 的应用。
59. 鉴定心室肌细胞调节因子的方法， 该方法包括：
1 )使权利要求 40或 51所述的心室肌细胞与候选的调节因子接触， 并测定该候选调节因子对心室肌细胞功能的影响；
2 ) 测定未与该候选调节因子接触的心室肌细胞的功能； 如果与该候选调节因子接触的心室肌细胞的功能较未与该候选 调节因子接触的心室肌细胞的功能不同，则确定该候选调节因子为所 述心室肌细胞的功能调节因子。
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CN104508121B (zh) 2018-01-19
JP6412868B2 (ja) 2018-10-24
JP2015527886A (ja) 2015-09-24
US20150299658A1 (en) 2015-10-22
EP2891712A1 (en) 2015-07-08
US20220340877A1 (en) 2022-10-27

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