Method for Inducing Pluripotent Stem Cells to Differentiate into Ventricular Cardiomyocytes In Vitro

Abstract

Provided in the present invention is a method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro, which is achieved by maintaining, amplifying and culturing pluripotent stem cells in vitro, adding a substance capable of activating the Smad1/5/8 signaling pathway directly or indirectly into the culture medium when pluripotent stem cells are in the middle stage of myocardial differentiation, i.e. the period of differentiating into cardiac muscle cells from mesoderm cells or myocardial precursor cells, which enables stem cells to differentiate into ventricular myocytes directionally. Ventricular myocytes with biological activity and function are obtained successfully by means of the method of the present invention, which reveals the regulatory mechanism during differentiation of myocardial precursor cells into ventricular myocytes; moreover, the human ventricular myocytes obtained via differentiation can be widely used in treating myocardial infarction by cell transplantation, in toxicological analysis of the heart and in the development of heart-related drugs.

Description

TECHNICAL FIELD

In certain aspects, the present invention relates to the fields of pluripotent stem cell (PSC) differentiation and signal transduction. In specific embodiments, the present invention involves methods to induce differentiation of PSCs into ventricular myocytes (VMs) in vitro.

BACKGROUND

In mammals, cardiomyocytes (CMs) are capable of cell division and proliferation before birth. However, their ability to proliferate rapidly declines after birth. Adult CMs typically have a very poor ability to proliferate. In heart diseases associated with cardiac tissue necrosis such as myocardial infarction, the consequent decline in cardiac function is typically irreversible because adult CMs have lost their ability to proliferate and are unable to repair necrotic tissue. Although medications can be used to increase cardiac contractility and improve the ability of the heart to pump blood, the heavier burden on the heart may in turn worsen the condition. Replacement of necrotic cells by transplantation of normal CMs is one of the methods for treatment of heart infarction and similar diseases or conditions. Because adult CMs have almost no ability to proliferate, a source of human CMs is apparently needed for regenerative medicine, for example, for treating myocardial infarction.

Pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). See, Thomson J A et al., Embryonic stem cell lines derived from human blastocysts, Science, 1998, 282:1145-1147; Yu J et al., Induced pluripotent stem cell lines derived from human somatic cells, Science, 2007, 318:1917-1920; and Takahashi K et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 2007, 131:861-872, the contents of which are incorporated herein by reference in their entireties. These pluripotent stem cells not only possess a strong ability to self-renew but also have the potentials for differentiation into CMs. Thus, PSC is one of the most promising cell sources of CMs if an efficient cardiac differentiation method is established.

In general, there are two methods to induce differentiation of CMs from PSCs. In one method, PSCs are cultured in suspension to form embryoid bodies that differentiate into numerous cell types including CMs. In the other method, monolayer PSCs under ordinary culture conditions are directly differentiated into CMs. A variety of cytokines have been reported to improve the efficiency of cardiac differentiation, and their dosages and duration of action vary based on the different differentiation systems.

Human PSC-derived CMs typically include three main types: nodal cells, VMs, and atrial myocytes (AMs). See, He J Q et al., Human embryonic stem cells develop into multiple types of cardiac myocytes: Action potential characterization, Circ Res. 2003, 93:32-39, the content of which is incorporated herein by reference in its entirety. According to their functional properties, fully mature CMs can be subdivided into working CMs and spontaneous beating nodal cells. Working CMs, including AMs and VMs that constitute the majority of the atrial and ventricular muscle walls, contain abundant myofibrils and possess the properties of conductivity and excitability, and perform systolic functions of the heart. Nodal cells spontaneously generate excitability, which controls the beating activity of heart. Similar to working CMs, the nodal cells possess the properties of conductivity and excitability, but typically have lost contractility. AMs, VMs, and nodal cells exhibit significant differences in the composition of intracellular myofibrils and cell membrane expression of ion channel proteins, resulting in substantial differences in their action potentials (APs) and their rhythmic contraction. For cell transplantation therapies for heart disease, it is essential to transplant cardiomyocytes with an appropriate subtype of high purity. For example, repairing ventricular tissue requires transplantation of VMs of high purity, which determines whether the cells can successfully integrate into the recipient heart tissue, improve heart functions, and reduce side effects such as arrhythmia caused by the transplanted cells. If the subtype of transplanted cardiomyocytes does not match the type of the tissue they are transplanted into, or the purity of transplanted CMs is not sufficient, arrhythmia may occur, impairing the function of the recipient heart. The left ventricle, which mainly carries the body blood supply, has the largest volume, thickest muscle walls, and strongest pumping capacity. Additionally, myocardial infarction occurs primarily in the left ventricle. Thus, among the three types of CMs, VMs are of the most significance for cell transplantation therapy of myocardial infarction. See, Chen H S et al., Electrophysiological challenges of cell-based myocardial repair, Circulation, 2009, 120:2496-250, the content of which is incorporated herein by reference in its entirety.

Obtaining large numbers of human CMs is important for the development of drugs for heart diseases, and the assessment of cardiac-toxicity of drugs. Adult human CMs cannot be expanded in vitro, leading to a lack of substantial numbers of human CMs for relevant experimental studies. Almost all cardiac-toxicological tests and experimental studies of drugs for heart disease are performed using animals or primary animal CMs. Owing to their physiological differences between human CMs and CMs from other animals, the accuracy of predicting a drug's effects on human using animals or their CMs is only about 60%. Thus, there are needs of improvement to the existing methods for heart related analysis in drug development. Human CMs derived from stem cells or trans-differentiation provide a tool for cardiac-toxicological analysis. The PSC derived human CMs can be used to establish methods for toxicological analysis at the cellular level. It not only improves the accuracy of the analysis, but also reduces the usage of animals. This approach is currently under extensively research in the bio-pharmaceutical industry. Relevant international regulations and provisions (ICH S7B) for drug registration require cardiac-toxicological analysis to assess the effects of tested drugs on the ventricle, especially the ventricular heart rhythm. Therefore, among the three types of CMs, VM is the most important subtype of CMs for the development of new methods for cardiac-toxicological analysis using human CMs. See, Hartung T, Toxicology for the twenty-first century, Nature, 2009, 460:208-212.

In summary, there is a need to generate highly homogeneous stem cell-derived human VMs for either cell transplantation therapy of myocardial infarction or cardiac-toxicological analysis. Therefore, revealing the regulatory mechanisms underlying differentiation of cardiac progenitor cells (CPCs) into VMs has significant implications for the generation of highly homogeneous VMs.

Previously reported methods for cardiac differentiation of stem cells have several drawbacks. The main issues are that the efficiency of cardiac differentiation is low, and the resulting CMs are a heterogeneous population of mixed nodal cells, AMs, and VMs. See, He J Q et al., Circ Res. 2003, 93:32-39. In 2007, Murry et al. used a monolayer culture of human ESCs to directly induce cardiac differentiation. The mean differentiation efficiency of the CMs was about 30%. See, Laflamme M A et al., Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts, Nat Biotechnol, 2007, 25:1015-1024, the content of which is incorporated herein by reference in its entirety. After separation and purification by density gradient centrifugation, the resulting CM population was approximately 80% in purity. In 2008, Keller et al. performed suspension culture to form embryoid bodies and then isolated CPCs at day 6 of differentiation by fluorescence-activated cell sorting. With continuous culture and differentiation of these progenitor cells, the cardiac differentiation efficiency was significantly improved, and reached up to 50%. See, Yang L et al., Human cardiovascular progenitor cells develop from a kdr+ embryonic-stem-cell-derived population, Nature, 2008, 453:524-528, the content of which is incorporated herein by reference in its entirety. However, none of the above methods can directly differentiate PSCs into highly homogeneous AMs or VMs. In summary, these methods do not facilitate the directed differentiation of AMs or VMs, and the cardiomyocytes population obtained using these methods is a mixture of all three types of CMs. At this point, there has been no relevant study on methods to specifically differentiate each of the three subtypes of CMs. In 2007, a study employed lentiviral transfection of human ESCs to establish a cell line expressing enhanced green fluorescent protein under the control of the conserved promoter of ventricle-specific myosin light chain 2v (MLC-2v) gene. This facilitated the purification of VMs and achieved a purity greater than 90%. However, this method requires insertion of a transgene into the genome of stem cells, and these transgenic CMs are unsuitable for clinical transplantation applications. In 2010, Ma et al. discovered that, during the middle stage of cardiac differentiation of stem cells, i.e., the period during which mesodermal cells convert to CMs (the early stage of cardiac differentiation refers to the stage of differentiation from PSCs to mesodermal cells), retinoic acid treatment induces differentiation of stem cells into AMs. At the same time, inhibition of the retinoic acid pathway effectively induces the cells to differentiate into VMs. See, Zhang Q et al., Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals, Cell Res., 2011, 21:579-587, the content of which is incorporated herein by reference in its entirety. However, the active regulators in the induction of ventricular differentiation remain unknown.

DESCRIPTION

In one aspect, the objective of the present invention is to provide methods to induce differentiation of PSCs into VMs in vitro.

To achieve this objective, in certain embodiments, the proposed method induces differentiation of PSCs into VMs by treating PSCs in vitro with factors that directly or indirectly activate the Smad1/5/8 signaling pathway during the middle stage of cardiac differentiation of PSCs, thereby achieving directed differentiation towards VMs. Here, in certain embodiments, activation of the Smad1/5/8 signaling pathway includes phosphorylation of one or more Smad proteins in the cytoplasm, including Smad1, Smad5, and Smad8. In certain embodiments, the middle stage of cardiac differentiation refers to the differentiation stage from mesodermal cells or cardiac progenitor cells to CMs. Specifically, in certain embodiments, this stage is initiated by expression of Brachyury (T) and/or Mespl genes, and ends before the differentiation of CMs capable of spontaneous contraction.

In any of the preceding embodiments, PSCs can include embryonic stem cells (ESC), induced pluripotent stem cells (iPSCs), embryonic germ cells, or adult stem cells. In any of the preceding embodiments, these cells can be from human or an animal.

In any of the preceding embodiments, the factors that directly or indirectly activate the Smad1/5/8 signaling pathway can be a bone morphogenetic protein, such as BMP 2 and/or BMP4 applied at a final concentration of between about 0.01 and about 1200 ng/mL.

In any of the preceding embodiments, the method can comprise adding one or more factors that promote differentiation of CMs during the early stage of cardiac differentiation of PSCs, namely the stage when PSCs differentiate into mesodermal or cardiac progenitor cells. In some embodiments, the factors that promote differentiation of CMs can include at least one of the following: BMP4, basic fibroblast growth factor (bFGF), activin A, noggin, dorsomorphin, and 6-bromoindirubin-3′-oxime (BIO), or a combination thereof. In any of the preceding embodiments, one or more growth factors can be added to the culture medium at a final concentration ranging from about 0.01 to about 1200 ng/mL. In any of the preceding embodiments, one or more small molecules can be added at a final concentration ranging from about 0.001 to about 100 μM.

In any of the preceding embodiments, one or more inhibitors of the Wnt signaling pathway can also be added to the culture medium during the middle stage of the cardiac differentiation. In any of the preceding embodiments, the Wnt inhibitors can include at least one of the following: dickkopf homolog 1 (DKK1), inhibitor of Wnt production (IWP), and inhibitor of Wnt response (IWR), or a combination thereof. In any of the preceding embodiments, the inhibitor can be added at a final concentration of about 0.001 to about 100 μM. In any of the preceding embodiments, DKK1 can be added with a final concentration from about 0.01 to about 1200 ng/mL.

In any of the preceding embodiments, one or more other regulatory molecules can also be added during the middle stage of cardiac differentiation, including: i) activator of retinoic acid receptor (RARγ) to culture medium without retinoic acid or its precursors; and ii) antagonists of RARα and/or RARβ to culture medium containing retinoic acid or its precursors. In any of the preceding embodiments, the final concentration of the regulatory molecule can be from about 0.001 to about 100 μM.

In some aspects, the present invention provides three technical solutions.

Technical solution I:

(1) Suspension or monolayer culture of undifferentiated PSCs.

(2) To initiate the cardiac differentiation, adding one or more cytokines (e.g., BMP4, bFGF, activin A, and noggin) that promote differentiation of CMs; and/or adding small molecule inhibitors (e.g., dorsomorphin) of the BMP pathway; and/or adding small molecules (e.g., BIO and CHIR99021) that activate Wnt3a signal pathway to the culture.

(3) During the middle stage of cardiac differentiation, adding one or more growth factors and/or small molecules (e.g., DKK1, IWP, and IWR) that inhibit the Wnt signaling pathway; and/or adding one or more signaling molecules (e.g., BMP2 and/or BMP4) that activate Smad1/5/8 phosphorylation to the culture medium, to induce directed differentiation of cells into VMs. In some embodiments, the inhibitor of the Wnt signaling pathway and the activator of Smad1/5/8 phosphorylation are added substantially simultaneously.

Technical solution II:

(1) Suspension or monolayer culture of undifferentiated PSCs.

(2) To initiate the cardiac differentiation, adding one or more cytokines (e.g., BMP4, bFGF, activin A, and noggin) that promote differentiation of CMs; and/or adding one or more small molecule inhibitors (e.g., dorsomorphin) of the BMP signaling pathway; and/or adding one or more small molecules (e.g., BIO) capable of activating Wnt3a signal pathway to the culture.

(3) During the middle stage of cardiac differentiation, adding one or more growth factors or small molecules (e.g., DKK1, IWP, and IWR) to the culture medium to inhibit the Wnt signaling pathway; and/or adding one or more factors that can activate cellular expression and secretion of signaling molecules which activate Smad1/5/8 phosphorylation. For example, a RARγ activator (e.g., BMS961) can be added to the culture medium without retinoic acid or its precursors or substrates (e.g., vitamin A). These steps induce directed differentiation of stem cells into VMs.

Technical solution III:

(1) Suspension or monolayer culture of substantially undifferentiated PSCs.

(2) To initiate the cardiac differentiation, adding one or more cytokines (e.g., BMP4, bFGF, activin A, and noggin) that promote differentiation of CMs; and/or adding one or more small molecule inhibitors (e.g., dorsomorphin) of the BMP pathway; and/or adding one or more small molecules (e.g., BIO) that activate Wnt3a signal pathway to the culture.

(3) During the middle stage of cardiac differentiation, adding one or more growth factors or small molecules (e.g., DKK1, IWP, and IWR) to the culture medium to inhibit the Wnt signaling pathway; and/or adding one or more antagonists of RARa and/or RARβ (e.g., Ro41-5253 for RARα and LE135 for RAM to the culture medium containing retinoic acid or its precursors. These steps induce directed differentiation of stem cell-derived CMs mainly into VMs.

In some embodiments, from about day 14 of differentiation, growth factor-free medium is used and replaced every 3 days. After about 60 to about 90 days of differentiation, the percentage of VMs in the differentiated CMs was determined with the analysis of APs of CMs (recorded by the patch clamp technique), calcium imagining studies, and/or MLC-2v and cardiac troponin T (cTNT) double immunofluorescence staining for flow cytometric analysis.

In some embodiments, the present invention enables the application of VMs prepared using a method of any of the preceding embodiments to the cardiac-toxicological analysis and drug screening for heart diseases.

In some embodiments, the present invention enables the application of VMs as prepared using a method of any of the preceding embodiments to stem cell therapy to repair damaged heart tissue.

In other embodiments, the present invention provides methods to promote stem cell differentiation into VMs. In some aspects, the method includes activation of the Smad1/5/8 signaling pathway in mesodermal cells that are derived from stem cells.

In any of the preceding embodiments, the stem cells can include totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and/or unipotent stem cells.

In any of the preceding embodiments, the stem cells can include ESCs, iPSCs, fetal stem cells, and/or adult stem cells.

In any of the preceding embodiments, the stem cells can include mammalian stem cells.

In any of the preceding embodiments, the stem cells can include human stem cells.

In any of the preceding embodiments, the stem cells can include human ESCs and/or iPSCs.

In any of the preceding embodiments, the stem cell differentiation into mesodermal cells can be induced by treating undifferentiated stem cells with at least one of the following: bFGF, BMP2, BMP4, activin A, a BMP antagonist, a BMP signaling pathway inhibitor, or a Wnt3a signaling pathway activator, or a combination thereof.

In any of the preceding embodiments, the BMP antagonist can be a BMP4 antagonist. In any of the preceding embodiments, the BMP antagonist can be noggin.

In any of the preceding embodiments, the BMP signaling pathway inhibitor can be a small molecule inhibitor of the BMP signaling pathway.

In any of the preceding embodiments, the small molecule inhibitor of the BMP signaling pathway can be dorsomorphin.

In any of the preceding embodiments, the Wnt3a signal pathway activator can be a small molecule activator of the Wnt3a signaling pathway.

In any of the preceding embodiments, the small molecule activator of the Wnt3a signaling pathway can be an ATP-competitive inhibitor of GSK-31303.

In any of the preceding embodiments, the ATP-competitive inhibitor of GSK-3α/β can be a cell-permeable bis-indolo (indirubin) compound.

In any of the preceding embodiments, the cell-permeable bis-indolo (indirubin) compound can be BIO.

In any of the preceding embodiments, bFGF, BMP2, BMP4, activin A, the BMP antagonist, the BMP signaling pathway inhibitor, and/or the Wnt3a signaling pathway activator, are added at a final concentration of about 0.01 to about 1200 ng/mL, whereas other factors can be added at a final concentration of about 0.001 to about 100 μM.

In any of the preceding embodiments, the stem cells can be treated with BMP2 and/or BMP4 to activate the Smad1/5/8 signaling pathway.

In any of the preceding embodiments, BMP2 and/or BMP4 can be applied at a final concentration of between about 0.01 and about 1200 ng/mL.

In any of the preceding embodiments, the stem cells can be cultured in a medium without retinoic acid or its precursors and treated with a RARγ activator to activate the Smad1/5/8 signaling pathway.

In any of the preceding embodiments, the precursor of retinoic acid can be vitamin A.

In any of the preceding embodiments, the RARγ activator can comprise BMS961, palovarotene, and/or CD437 (e.g., purchased from Sigma-Aldrich).

In any of the preceding embodiments, the RARγ activator can be applied at a final concentration of about 0.001 to about 100 μM.

In any of the preceding embodiments, the stem cells can be treated with one or more RARα and/or RARβ antagonists to activate the Smad1/5/8 signaling pathway.

In any of the preceding embodiments, the RARα antagonist can be Ro41-5253, BMS195614, or ER50891, and the RARβ antagonist can be LE135.

In any of the preceding embodiments, the antagonist of RARα and/or RARβ can be applied at a final concentration of about 0.001 to about 100 μM.

In any of the preceding embodiments, the stem cells can be treated further with a Wnt inhibitor to induce differentiation into VMs.

In any of the preceding embodiments, the Wnt inhibitor can be at least one of the following: DKK1, IWP, or IWR.

In any of the preceding embodiments, the Wnt inhibitor DKK1 can be used at a final concentration of about 0.01 to about 1200 ng/mL, while other inhibitors can be used at about 0.001 to about 100 μM.

In some aspects, the present invention discloses VMs generated by following the method in any of the preceding embodiments.

In any of the preceding embodiments, the VMs can have increased levels or ratios of ventricular-specific gene expression, embryonic ventricular-like APs, and/or the representative characteristic of VMs specific Ca²⁺ activity (e.g., Ca′ spark).

In any of the preceding embodiments, the ventricle-specific gene can be Iroquois homeobox gene 4 (IRX-4) and/or MLC-2v.

In other embodiments, the present invention provides a composition containing stem cells that have differentiated into mesodermal cells and have been treated with an exogenous factor capable of activating the Smad1/5/8 signaling pathway in stem cells.

In any of the preceding embodiments, the exogenous factor that activates the Smad1/5/8 signaling pathway in stem cells can be BMP2 and/or BMP4.

In any of the preceding embodiments, the exogenous factor that activates the Smad1/5/8 signaling pathway in stem cells can be a RARγ activator.

In any of the preceding embodiments, the exogenous factor that activates the Smad1/5/8 signaling pathway in stem cells can be a RARα and/or RARβ antagonist.

In yet other embodiments, the present invention provides a method of deriving VMs from stem cells, which method comprises: 1) treating stem cells with bFGF and BMP4 to induce differentiation; 2) exposing bFGF and BMP4-treated stem cells to activin A to induce mesodermal cells; 3) treating stem cells that have been differentiated into mesoderm cells with noggin to improve the efficiency of stem cell differentiation towards CMs; 4) activating the Smad1/5/8 signaling pathway in noggin-treated stem cells to promote the differentiation of VMs; and 5) exposing noggin-treated stem cells to one or more factors to induce stem cell differentiation into VMs. In some aspects, the one or more factors comprise at least one of the following: DKK1, IWP, and IWR.

In any of the preceding embodiments, the stem cells can be treated with BMP2 and/or BMP4 to activate the Smad1/5/8 signaling pathway.

In any of the preceding embodiments, the stem cells can be cultured in medium without retinoic acid or its precursor, vitamin A, and the cultured cells can be treated with one or more RARγ activators to increase BMP2/4 expression levels to activate the Smad1/5/8 signaling pathway.

In any of the preceding embodiments, the stem cells can be treated with RARa and/or RARβ antagonists to activate the Smad1/5/8 signaling pathway.

In other aspects, the present invention discloses VMs generated by the method in any of the preceding embodiments.

In some aspects, the present invention provides a pharmaceutical composition for treating a subject in need thereof, for example, a subject with heart damage or disease, which composition comprising, consisting essentially of, or consisting of an effective amount of the VMs according to any of the preceding embodiments, and a pharmaceutically acceptable carrier or excipient. In some aspects, provided herein is a method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the preceding embodiments. In some aspects, provided herein is a method comprising administering to a subject in need thereof an effective amount of the VMs according to any of the preceding embodiments.

In any of the preceding embodiments, the subject can be a human.

In any of the preceding embodiments, the method can be used in a stem cell therapy of a heart damage, disease, or condition.

In some embodiments, the present invention provides the use of the VMs according to any of the preceding embodiments for the preparation or manufacture of a medicament for the treatment and/or prevention of a heart damage, disease, or condition.

In some embodiments, the present invention also provides the use of the VMs according to any of the preceding embodiments for screening and/or developing drugs for the treatment and/or prevention of a heart damage, disease, or condition.

In other embodiments, the present invention provides the use of the VMs according to any of the preceding embodiments for cardiac-toxicological analysis for drug safety.

In still other embodiments, the present invention provides a method to identify regulators of VMs by a) treating a VM according to any of the preceding embodiments with one or more candidate regulators and determining the effect of the candidate regulator on the function of VM, and 2) determining the function of VM without treatment with the candidate regulator. If the function of the VM treated with the candidate regulator differs from that of the VM without treatment with the candidate regulator, the candidate regulator is identified as a functional regulator of VMs.

In some embodiments, the present invention provides a method to induce differentiation of PSCs into VMs in vitro. In some aspects, based on direct induction of stem cells, factors (e.g., BMP2 and/or BMP4) that can activate the Smad1/5/8 signaling pathway are directly added to the culture system during the middle stage of cardiac differentiation, thereby directing differentiation of stem cells into CMs. In some aspects, the CMs are mainly VMs. In the culture system containing retinoic acid or its precursors (e.g., vitamin A), addition of factor(s) (e.g., BMP2 and/or BMP4) capable of activating the Smad1/5/8 signaling pathway can effectively inhibit the differentiation of CPCs to AMs while inducing the differentiation towards VMs. In some embodiments, when retinoic acid or vitamin A are added simultaneously with such factor(s) (e.g., BMP2 and/or BMP4) capable of activating the Smad1/5/8 signaling pathway, the proportion of AMs among differentiated CMs decreases with increasing concentrations of BMP4, whereas the proportion of VMs among differentiated CMs increases with increasing BMP4 concentrations. In some aspects, when BMP2/4 only is added during the middle stage of cardiac differentiation when retinoic acid or its precursors are omitted from the culture medium, cardiac progenitor cells efficiently undergo directed differentiation into VMs. In some aspects, during the middle stage of cardiac differentiation of stem cells, addition of an activator of RARα or RARβ to the culture medium effectively inhibits the differentiation of VMs, as reflected by the inhibition of ventricle-specific expression of the early marker gene IRX-4. In some aspects, in the absence of retinoic acid or its precursor vitamin A, addition of a RARγ activator effectively induces stem cell differentiation into VMs. In some aspects, in culture medium containing retinoic acid or vitamin A, addition of RARα and RARβ antagonists is effective to improve the IRX-4 expression level and induce stem cell differentiation into VMs.

In some aspects, the present invention illustrates that BMP and Smad1/5/8 pathways positively regulate differentiation of VMs during the middle stage of cardiac differentiation of stem cells. In some aspects, the present invention allows the generation of highly homogeneous VMs with a desirable biological activity or function.

In some aspects, as an advantage, the present invention does not require any purification steps. In some other aspects, the present invention provides a platform to reveal the regulatory mechanisms underlying differentiation of cardiac progenitor cells to VMs. In still other aspects, the present invention has significant implications for cell transplantation therapy of myocardial infarction as well as drug research and development using human stem cell-derived CMs.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show the expression of molecules involved in the BMP signaling pathway during the middle stage of cardiac differentiation of stem cells, and the effects on expression of the ventricle-specific marker gene IRX-4. FIG. 1A shows reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression of BMP2, BMP4, and their receptors at 5 and 6 days of cardiac differentiation. FIG. 1B shows western blot analysis of downstream signaling molecules [phosphorylated Smad1/5/8 (P-Smad1/5/8)] of the BMP pathway. T-Smad1/5/8 represents total Smad1/5/8 proteins. (3-actin served as an internal loading control. The histogram in FIG. 1C presents the experimental results of quantitative RT-PCR analysis of IRX-4 gene expression levels at 14 days of differentiation. The results show IRX-4 expression levels in cells treated with 1 μM retinoic acid and 200 ng/mL BMP4 during different stages of differentiation. Connected line indicates the cardiac differentiation efficiency of the stem cells under the corresponding inductive conditions. N represents noggin, B represents BMP4; NVa represents differentiated cells cultured in vitamin A-free medium; RA represents retinoic acid; and numbers represent the concentrations (Unit for BMP4 is ng/mL). Data are expressed as relative values compared with the expression level of glyceraldehyde-3-phosphate dehydrogenase (GADPH).

FIGS. 2A-2D present quantitative RT-PCR analysis of the expression levels of the ventricle-specific early marker gene IRX-4 at day 14 in differentiated cultures with various treatments. FIG. 2A shows that, after the addition of BMP4 with various concentrations to the cultures, the IRX-4 expression level is elevated with increasing concentrations of BMP4. However, the expression level is reduced by addition of a BMP antagonist, noggin. FIG. 2B illustrates that the IRX-4 expression level is effectively reduced by addition of various doses of noggin to the medium without the retinoic acid precursor, vitamin A. FIG. 2C shows that in the presence of 1 μM retinoic acid, the IRX-4 expression level is elevated with increasing concentrations of BMP4 added to the cultures. FIG. 2D shows that the elevation of the IRX-4 expression level by BMP4 in the presence of retinoic acid is reduced with additions of increasing concentrations of noggin in the cultures. N represents noggin; B represents BMP4, NVa represents differentiated cells treated in vitamin A-free medium; RA represents retinoic acid; and numbers represents the concentrations (Unit is ng/mL). The results of quantitative RT-PCR are indicated as relative values compared with the expression levels of GADPH.

FIG. 3 presents quantitative RT-PCR analysis of IRX-4 gene expression levels at day 14 of differentiation. The results show that by treatment of the stem cells at days 5-8 of cardiac differentiation, other members of the BMP family also effectively antagonized the inhibitory effect of retinoic acid on IRX-4 expression. The antagonistic effect is enhanced with increasing doses of BMP family member growth factors. RA represents retinoic acid; numbers represent the concentrations of the growth factor (Unit is ng/mL); the concentration of retinoic acid is 1 μM. The results of quantitative RT-PCR are indicated as relative values compared with the expression levels of GADPH.

FIGS. 4A-4B display ventricle-specific MLC-2v expression in long term cultures that differently treated with retinoic acid, BMP4, and noggin. FIG. 4A shows western blot analysis of MLC-2v expression in stem cell-derived CMs at day 90 of differentiation treated with retinoic acid, noggin and BMP4 with different combinations. FIG. 4B presents the results of double immunofluorescence staining of cTNT and MLC-2v in CMs at day 90 of differentiation after retinoic acid, BMP4, and noggin treatments. Letter B in the figures represents BMP4, NVa represents differentiated cells cultured in vitamin A-free medium; RA represents 1 μM retinoic acid; and numbers represent the concentrations (ng/mL).

FIGS. 5A-5B present images from confocal laser scanning microscopy and simultaneous recordings of APs of calcium activity in differentiated CMs and the classification of the differentiated CMs according to the specific calcium activity patterns in the various types of CMs. FIG. 5A shows the features of Ca²⁺ sparks in CMs with ventricular-like APs, Ca′ transients in cells with atrial-like APs, and Ca²⁺ oscillations in cells with nodal-like APs. FIG. 5B presents the proportions of CMs with Ca²⁺ sparks, Ca²⁺ transients, and Ca²⁺ oscillations in different treatments as classified by calcium signaling patterns of the various subtypes of CMs in A. The vertical axis represents the proportions of CMs with the three different calcium activities; RA represents 1 μM retinoic acid; Letter B represents BMP4; NVa represents vitamin A-free medium; N represents noggin; and numbers represent the concentrations (ng/mL).

FIG. 6 shows quantitative RT-PCR analysis of BMP2 expression in cells treated with a RARγ activator at day 6 of differentiation. The results of quantitative RT-PCR are indicated as relative values compared with those of GADPH. NVa represents vitamin A-free medium.

FIG. 7 shows quantitative RT-PCR analysis of ventricle-specific IRX-4 expression at day 14 of stem cell differentiation after the addition of various regulators (a RAR activator or inhibitor) to vitamin A-free medium during middle stage of cardiac differentiation of stem cells. RA represents retinoic acid; RAi represents a retinoic acid inhibitor, BMS189453; NVa represents vitamin A-free medium; The RAR pan-antagonist is BMS493. The results of quantitative RT-PCR are expressed as relative values compared with cTNT expression

FIGS. 8A-8B show the proportions of cardiomyocytes with different AP characteristics, and MLC-2v (a mature VM-specific marker gene) expressing cells in the total cardiomyocyte population differentiated under various differentiation conditions. FIG. 8A shows the proportion of cells with atrial-, ventricular-, and nodal-like APs in CMs differentiated under various induction conditions (n>30).

FIG. 8B shows flow cytometric analyses of the proportions of MLC-2v-expressing cells in the total cardiomyocytes population (cTNT-positive cells) among 90 day cultures treated under various conditions. RA represents 1 μM retinoic acid; B represents BMP4; NVa represents vitamin A-free medium; N represents noggin; and numbers represent the concentrations (Unit is ng/mL). The RARγ concentration is 0.1 μM.

FIG. 9 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 2 (infra) of the present invention.

FIG. 10 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 3 (infra) of the present invention.

FIG. 11 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 4 (infra) of the present invention.

FIG. 12 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after retinoic acid treatment.

FIG. 13 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with retinoic acid and 200 ng/mL BMP4.

FIG. 14 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with 1200 ng/mL noggin in vitamin A-free medium.

FIG. 15 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 2 (infra).

FIG. 16 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after culture in vitamin A-free medium.

FIG. 17 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 3 (infra).

In FIGS. 12-17, “*” indicates non-ventricular CMs and “{circumflex over ( )}” indicates MLC-2v-expressing VMs.

EXAMPLES

The following examples are provided to describe the present invention, but do not restrict the scope of the present invention. Unless specified otherwise, the technical terms used in the embodiments are conventional terms known to those individuals skilled in the procedures using materials that are commercially available.

In the following examples, the human ESC line H7 was purchased from WiCell Research Institute, USA; B27 supplement and RPMI1640 medium were purchased from Invitrogen; Activin A, bFGF, DKK1, BMP4, and noggin were purchased from R&D systems.

Example 1: Role of BMP/Smad1/5/8 Signaling Pathways in Inducing Differentiation of Cardiac Progenitor Cells into VMs

1) Previous research indicated that during cardiac differentiation of stem cells, addition of retinoic acid or its precursors (e.g., vitamin A) to the culture medium at the differentiation stage that determines the subtype of CMs induces directed differentiation of stem cells into AMs. On the other side, addition of a retinoic acid inhibitor to the culture medium or exclusion of vitamin A in the culture medium induces directed differentiation of stem cells into VMs. See, Zhang Q et al., Cell Res., 2011, 21:579-587. Using RT-PCR, the expression of BMP2/4 and their corresponding receptor in differentiated human ESCs was analyzed during the middle stage of cardiac differentiation. As shown in FIGS. 1A-1C, both the ligands and receptors of the BMP pathway are expressed in the cultured cells. Western blot analysis of BMP2/4 downstream signaling molecules (phosphorylated Smad1/5/8) showed that under the culture condition with retinoic acid addition and in the absence of retinoic acid or vitamin A, Smad1/5/8 molecules are phosphorylated (FIGS. 1A-1C). These results demonstrated that activation of the BMP pathway during the middle stage of cardiac differentiation of stem cells. These results indicated that during days 5-8 of stem cell differentiation, the BMP pathway is involved in regulating the differentiation of cardiac progenitor cells into VMs.

2) The role of the BMP pathway in directed differentiation of CM subtypes was analyzed further during cardiac differentiation of stem cells. IRX-4 is a marker gene expressed during early differentiation of VMs. Thus, the IRX-4 expression level was measured to further study the role of the BMP pathway in differentiation of CM subtypes.

As indicated in FIGS. 2A-2D, in vitamin A-free medium, the IRX-4 expression level was effectively reduced by addition of a BMP2/4 pathway inhibitor, noggin, during day 5 to day 8 of differentiation. The expression level of IRX-4 decreased with increasing doses of noggin (300, 600, and 1200 ng/mL).

3) IRX-4 expression level was repressed by addition of retinoic acid during day 5 to day 8 of differentiation. Furthermore, when retinoic acid was added simultaneously with various doses of BMP4 during day 5 to day 8 of differentiation, and the measurement of the expression levels of IRX-4 by quantitative RT-PCR showed that the IRX-4 expression level in retinoic acid-treated culture was elevated by addition of BMP. As the dose of BMP4 increased, the expression level of IRX-4 increased correspondingly (FIGS. 2A-2D).

Additionally, other members of the BMP family antagonize the inhibitory effect of retinoic acid on IRX-4 expression. Most BMP family members have similar functions. Quantitative RT-PCR analysis (FIG. 3) showed that during middle stage of cardiac differentiation, the IRX-4 expression level was elevated to various degrees by other BMP family members in the presence of 1 μM retinoic acid. The expression level of IRX-4 increased with increasing concentrations of those BMP family members added to the medium.

In summary, the analysis of early specific IRX-4 expression in differentiated cultures indicated that the BMP signaling pathway effectively improves IRX-4 expression in differentiated CMs. This finding shows that during differentiation of stem cells, the BMP signaling pathway is involved in their early differentiation into VMs and plays a role in promoting this process.

4) To verify the role of the BMP pathway in regulating the differentiation of VMs from stem cells, calcium activities of cardiomyocytes were recorded with confocal laser scanning microscopy at 60-90 days of differentiation. Calcium activity in VMs clearly differs from that in AMs and nodel cells. Calcium activity in VMs has a higher imaging frequency, known as Ca⁺ sparks. Imaging of the AM calcium activity showed a lower frequency with large signals, called Ca⁺ transients, whereas imaging of calcium activity in nodel cells demonstrated obvious periodicity called Ca⁺ oscillations. First, using the single-cell patch clamp technique in conjunction with confocal laser scanning microscopy, it was found that patch clamp-recorded calcium activities in 20 cells with ventricular-like APs exclusively shows Ca⁺ sparks. Patch clamp-recorded calcium activities in 20 cells with atrial-like APs exclusively showed Ca⁺ transients, whereas patch clamp-recorded calcium activities in 20 cells with nodal-like APs exclusively shows Ca⁺ oscillations. Thus, comparing the image pattern of calcium activities in CMs is an effective method to distinguish VMs from AMs and nodel cells. Calcium imaging data showed that the majority of differentiated CMs with retinoic acid treatment had Ca⁺ transients, while the proportion of cells with Ca⁺ transients decreased with increasing concentrations of BMP4. In contrast, the proportion of cells with Ca⁺ sparks among differentiated CMs increased with increasing concentrations of BMP4. This result indicates that activation of the BMP signaling pathway effectively induces stem cells to differentiate into VMs (FIGS. 5A-5B).

5) As mentioned above, during early differentiation of CMs, IRX-4 is an important gene with specific expression in differentiated VMs. As CMs mature, VMs begin to specifically express the MLC-2v gene. Thus, the MLC-2v expression level was measured in differentiated cells at day 90 of culture after treatment with various growth factors. Western blot analysis showed that the MLC-2v protein expression level in differentiated cells at day 90 also increased with increasing concentrations of BMP4 (FIGS. 4A-4B). Moreover, flow cytometry was performed to determine the proportion of MLC-2v-expressing VMs among differentiated CMs (cTNT-expressing cells) at day 90. The results (FIGS. 8A-8B) showed that addition of BMP4 to differentiated cells after retinoic acid treatment effectively increased the proportion of MLC-2v-expressing cells among differentiated CMs, with the highest proportion obtained by BMP4 treatment alone. The most classical method to identify the subtypes of CMs is to measure the APs of the CMs. The proportions of cells with atrial-, ventricular-, and nodal-like APs among differentiated CMs were analyzed after treatment with retinoic acid and various doses of BMP4 (FIGS. 8A-8B). Among differentiated CMs after retinoic acid treatment, the proportion of cells with ventricular-like APs increased significantly with increasing doses of BMP4. Among differentiated cells treated with BMP4 alone, more than 90% of CMs had ventricular-like APs, indicating that more than 90% of CMs were VMs.

6) Addition of BMP2/4, activin A, bFGF and/or noggin during early cardiac differentiation of ESCs and growth factors such as DKK1 during the middle stage of cardiac differentiation efficiently induced stem cells to differentiate into CMs. Quantitative RT-PCR analysis showed that ventricle-specific IRX-4 expression was reduced by addition of RARα and RARβ activators along with DKK1 during middle stage of cardiac differentiation (FIG. 7). However, high expression of BMP2 and IRX-4 was induced by addition of DKK1 with a RARγ activator (FIGS. 6 and 7). Additionally, in vitamin A-containing medium, early specific IRX-4 expression in VMs was activated by addition of DKK1 and antagonists of RARα and RAR. This indicated induced differentiation of stem cells into VMs. Because retinoic acid has three RARs receptors (RARα, RARβ, and RARγ), simultaneous inhibition of RARα and RARβ in the presence of vitamin A or retinoic acid has a similar mechanism and effect as that of independent activation of RARγ alone.

Flow cytometric analysis (FIGS. 8A-8B) demonstrated that the proportion of MLC-2v-expressing cardiomyocytes in cardiomyocytes population (cTNT-expressing cells) reached up to 80% at 90 days of differentiation induced by RARγ. Additionally, electrophysiological identification of APs indicated that 92% of RARγ-induced, differentiated CMs at 90 days had ventricular-like APs.

Example 2: Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution I)

The human ESC line H7 was cultured on gelatin-coated petri dishes in RPMI 1640 medium supplemented with B27 at 37° C. in a CO₂ incubator. The process of cardiac differentiation is presented in FIG. 9. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with a BMP2/4 inhibitor, noggin (300 ng/mL) added to the medium. At the end of day 5, the medium was replaced with vitamin A-free, B27 supplemented RPMI1640 medium. A Wnt3a inhibitor, DKK1 (300 ng/mL) and BMP4 (10 ng/mL) were also added to the medium. At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 9.

Example 3: Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution II)

The human ESC line H7 was cultured on gelatin-coated petri dishes in RPMI 1640 medium with 1×B27 at 37° C. in a CO₂ incubator. The process of cardiac differentiation is presented in FIG. 10. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with differentiation medium containing a BMP2/4 inhibitor, noggin (300 ng/mL). At the end of day 5, the medium was replaced with vitamin A-free, B27-containing RPMI 1640 medium containing a Wnt3a inhibitor, DKK1 (300 ng/mL), and RARγ activator, BMS961 (0.1 μM, Tocris). At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI 1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 10.

Example 4: Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution III)

The human ESC line H7 was cultured on gelatin-coated petri dishes in 1×B27-containing RPMI 1640 medium at 37° C. in a CO₂ incubator. The process of cardiac differentiation is presented in FIG. 11. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with differentiation medium containing a BMP2/4 inhibitor, noggin (300 ng/mL). At the end of day 5, the medium was replaced with vitamin A-free, B27-containing RPMI1640 medium containing a Wnt3a inhibitor, DKK1 (300 ng/mL), as well as antagonists of RARα and RARβ, BMS195614 (0.1 μM) and LE135 (0.5 μM), respectively. At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the culture medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI 1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 11.

FIG. 12 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after retinoic acid treatment.

FIG. 13 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with retinoic acid and 200 ng/mL BMP4.

FIG. 14 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with 1200 ng/mL Noggin in vitamin A-free medium.

FIG. 15 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 2.

FIG. 16 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after culture in vitamin A-free medium.

FIG. 17 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 3.

In FIGS. 12-17, “*” indicates non-ventricular CMs and^“A” indicates MLC-2v-expressing VMs.

Confocal laser scanning microscopy was performed to analyze calcium activities in differentiated CMs acquired from Example 2 and 3 at 60-90 days. The results of calcium imaging are shown in FIGS. 5A-5B, from which the proportion of cells with Ca²⁺ sparks among total differentiated CMs can be calculated directly.

In the above examples, the effective ranges of the final concentration for the relevant additives in the medium are 0.01-1200 ng/mL for growth factors and 0.001-100 μM for small molecules.

In some aspects, the methods disclosed in the present invention successfully generate biologically active and functional VMs. These methods can be used to reveal the regulatory mechanisms of CPC differentiation into VMs, whereas the resulting differentiated human VMs have extensive applications in cell transplantation therapy of myocardial infarction, toxicological analysis of cardiac drugs, and cardiac drug development.

Although the present invention has been fully described with general instructions and specific embodiments, it is noted that various changes and modifications will become apparent to those skilled in the procedures. Therefore, such changes and modifications made to the invention without departing from its essence are being protected within the scope of the present invention as claimed.

INDUSTRIAL APPLICABILITY

In some aspects, the present invention provides a method to induce differentiation of PSCs into VMs in vitro, which successfully generates biologically active and functional VMs. It can not only reveal the regulatory mechanisms underlying differentiation of VMs from CSCs, but also produce human VMs that have broad applications in cell transplantation therapy of myocardial infarction, as well as cardiac-toxicological analysis of drug safety, and drug development for heart diseases.

Claims

1-11. (canceled)

12. A method for promoting pluripotent stem cell differentiation into ventricular cardiomyocyte(s), the method comprises activating the Smad1/5/8 signaling pathway in a pluripotent stem cell at the middle stage of pluripotent stem cell differentiation, wherein the middle stage is when a mesodermal cell or a cardiac progenitor cell differentiates into a cardiomyocyte.

13. The method of claim 12, characterized in that the stem cell is a pluripotent stem cell, a totipotent stem cell, a multipotent stem cell, an oligopotent stem cell, or a unipotent stem cell.

14. The method of claim 12, characterized in that the stem cell is an embryonic stem cell, an induced pluripotent stem cell, a fetal stem cell, or an adult stem cell.

15. The method of claim 12, characterized in that the stem cell is a mammalian stem cell.

16. The method of claim 15, characterized in that the mammalian stem cell is a human stem cell.

17. The method of claim 12, characterized in that the stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

18. The method of claim 12, characterized in that the stem cell has differentiated to form mesoderm by contacting an undifferentiated stem cell with one or more of basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP 2), bone morphogenetic protein 4 (BMP 4), activin A, a BMP antagonist, a BMP pathway inhibitor, and a Wnt3a pathway activator.

19-27. (canceled)

28. The method of claim 12, characterized in that the Smad1/5/8 pathway is activated by contacting the stem cell with BMP 2 and/or BMP 4.

29. The method of claim 28, characterized in that the BMP 2 and/or BMP 4 is used at a final concentration of 0.01-1200 ng/ml.

30. The method of claim 12, characterized in that the Smad1/5/8 pathway is activated by culturing the stem cell in a medium that does not comprise retinoic acid or its precursor and contacting the stem cell with an agonist of retinoic acid receptor 7 (RARγ).

31. The method of claim 30, characterized in that the retinoic acid precursor is vitamin A.

32. The method of claim 30, characterized in that the RARγ agonist is BMS961, Palovarotene, or CD437 from SIGMA-ALDRICH.

33. The method of claim 30, characterized in that the RARγ agonist is used at a final concentration of 0.001-100 μM.

34. The method of claim 12, characterized in that the Smad1/5/8 pathway is activated by contacting the stem cell with an antagonist of retinoic acid receptor α (RARα) and/or retinoic acid receptor β (RARβ).

35. The method of claim 34, characterized in that the antagonist of RARα is Ro41-5253, BMS195614, or ER50891, and the antagonist of RARβ is LE135.

36. The method of claim 33, characterized in that the antagonist of RARα and/or RARβ is used at a final concentration of 0.001-100 μM.

37-51. (canceled)

52. A pharmaceutical composition for treating a cardiac injury or disorder, which pharmaceutical composition comprises an effective amount of the ventricular cardiomyocytes produced by the method of claim 12, and a pharmaceutically acceptable carrier or excipient.

53. A method for treating a cardiac injury or disorder in a subject, which method comprises administering, to a subject to which such treatment is needed or desirable, an effective amount of the pharmaceutical composition of claim 52.

54-59. (canceled)