# Patent

## Title
Methods and materials for providing cardiac cells

## Abstract

This document provides methods and materials relating to cardiac cells. For example, this document provides methods and materials that can be used to obtain cells having the ability to differentiate into cardiomyocytes. Such cells can be used to repair damaged heart tissue. For example, cells having the ability to differentiate into cardiomyocytes can be used to repair or regenerate heart tissue in patients with a cardiac condition (e.g., ischemic cardiomyopathy, myocardial infarction, or heart failure).

## Background
CLAIM OF PRIORITY
This application claims priority under 35 U.S.C. §119(e) to U.S. Patent Application Ser. No. 60/832,845, filed on Jul. 24, 2006, which is hereby incorporated by reference in its entirety.


BACKGROUND
1. Technical Field
This document relates to methods and materials involved in obtaining cardiac cells. For example, this document relates to methods and materials for providing mammalian heart tissue with cells that differentiate into cardiomyocytes.
2. Background Information
Cardiovascular disease is a leading cause of morbidity and mortality worldwide, despite advances in patient management (Towbin and Bowles, Nature, 415:227-233 (2002)). In contrast to tissues with high reparative capacity, heart tissue is vulnerable to irreparable damage (Anversa and Nadal-Ginard, Nature, 415:240-243 (2002)). Cell-based regenerative cardiovascular medicine is, therefore, being pursued in the clinical setting (Dimmeler et al., J Clin Invest, 115:572-583 (2005); Wollert and Drexler, Circ Res, 96:151-163 (2005); Caplice et al., Nat Clin Pract Cardiovasc Med, 2:37-43 (2005)).
SUMMARY
This document provides methods and materials relating to cardiac cells. For example, this document provides methods and materials that can be used to obtain cells having the ability to differentiate into cardiomyocytes. Such cells can be used to repair damaged heart tissue. For example, cells having the ability to differentiate into cardiomyocytes can be used to repair or regenerate heart tissue in patients with a cardiac condition (e.g., ischemic cardiomyopathy, myocardial infarction, or heart failure).
In general, one aspect of this document features a cell having the ability to differentiate into a cardiomyocyte, wherein the cell comprises an Nkx2.5 polypeptide and a MEF2C polypeptide associated with the nucleus of the cell. The DNA structure of the cell can be such that it is not modified through demethylation or histone deacetylation.
In another aspect, this document features a cell having the ability to differentiate into a cardiomyocyte, wherein the cell comprises an Nkx2.5 polypeptide, a MEF2C polypeptide, and a GATA4 polypeptide associated with the nucleus of the cell. The DNA structure of the cell can be such that it is not modified through demethylation or histone deacetylation.
In another aspect, this document features a cell having the ability to differentiate into a cardiomyocyte, wherein the cell is obtained by a method comprising contacting a stem cell with a composition under conditions wherein the stem cell differentiates into the cell, wherein the composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that the composition comprises BMP, α-thrombin, or TNF-α when the composition comprises less than six of the molecules of the group. The cell can comprise a Nkx2.5 polypeptide, a MEF2C polypeptide, and a GATA4 polypeptide associated with the nucleus of the cell. The cell can maintain the ability to differentiate into a cardiomyocyte for 10 cell divisions if the cell is contacted with the composition for two days and the composition is removed from the cell after two days. The cell can maintain the ability to differentiate into a cardiomyocyte for 10 cell divisions if the cell is contacted with the composition for five days and the composition is removed from the cell after five days. The cell can form a sarcomere if the cell is contacted with the composition for 15 days. The cell can produce a calcium transient in response to an electrical current if the cell is contacted with the composition for 21 days. The cardiomyocyte can be a human cardiomyocyte. The stem cell can express CD105, CD166, CD29, and CD44 polypeptides and can lack expression of CD14, CD34, and CD45 polypeptides. The stem cell can be a mesenchymal stem cell. The stem cell can be a human mesenchymal stem cell. The stem cell can be obtained from human bone marrow. The at least one of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. Each of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. The composition can comprise between 2.5 ng per mL and 10 ng per mL of the TGF-β. The composition can comprise between 5 ng per mL and 20 ng per mL of the BMP. The composition can comprise between 5 ng per mL and 50 ng per mL of the TNF-α. The composition can comprise between 1×10−6 μM and 2×10−6 μM of the retinoic acid. The composition can comprise between 50 ng per mL and 100 ng per mL of IGF-1, between 10 ng per mL and 20 ng per mL of FGF-4, between 100 ng per mL and 200 ng per mL of IL-6, between 5 ng per mL and 200 ng per mL of VEGF-A, and 40 nM of α-thrombin. The composition can comprise BMP, α-thrombin, and TNF-α when the composition comprises less than six of the molecules of the group.
In another aspect, this document features a method for providing heart tissue with cardiomyocytes, wherein the method comprises administering, to the heart tissue, cells comprising Nkx2.5 polypeptides and MEF2C polypeptides associated with the nuclei of the cells. The cardiomyocytes can be human cardiomyocytes. The cells can be obtained by contacting stem cells with a composition under conditions wherein the stem cells differentiate into the cells, wherein the composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that the composition comprises BMP, α-thrombin, or TNF-α when the composition comprises less than six of the molecules of the group. The stem cells can express CD105, CD166, CD29, and CD44 polypeptides, and can lack expression of CD14, CD34, and CD45 polypeptides. The stem cells can be mesenchymal stem cells. The stem cells can be human mesenchymal stem cells. The stem cells can be obtained from human bone marrow. The at least one of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. Each of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. The cell can maintain capacity for proliferation, capacity for nuclear translocation of cardiac transcription factors, capacity for sarcomeric organization, and capacity for calcium transient/contractile response to electrical stimulation provided by the heart tissue. The cell can have functional excitation contraction coupling.
In another aspect, this document features a method for providing heart tissue with cardiomyocytes, wherein the method comprises administering, to the heart tissue, cells comprising Nkx2.5 polypeptides, MEF2C polypeptides, and GATA4 polypeptides associated with the nuclei of the cells. The cardiomyocytes can be human cardiomyocytes. The cells can be obtained by contacting stem cells with a composition under conditions wherein the stem cells differentiate into the cells, wherein the composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that the composition comprises BMP, α-thrombin, or TNF-α when the composition comprises less than six of the molecules of the group. The stem cells can express CD105, CD166, CD29, and CD44 polypeptides, and can lack expression of CD14, CD34, and CD45 polypeptides. The stem cells can be mesenchymal stem cells. The stem cells can be human mesenchymal stem cells. The stem cells can be obtained from human bone marrow. The at least one of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. Each of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. The cell can maintain capacity for proliferation, capacity for nuclear translocation of cardiac transcription factors, capacity for sarcomeric organization, and capacity for calcium transient/contractile response to electrical stimulation provided by the heart tissue. The cell can have functional excitation contraction coupling.
In another aspect, this document features a composition comprising TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, wherein the composition comprises bovine serum albumin at 10−6 μM. The at least one of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. Each of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. The composition can comprise between 2.5 ng per mL and 10 ng per mL of the TGF-β. The composition can comprise between 5 ng per mL and 20 ng per mL of the BMP. The composition can comprise between 5 ng per mL and 50 ng per mL of the TNF-α. The composition can comprise between 1×10−6 μM and 2×10−6 μM of the retinoic acid. The composition can comprise between 50 ng per mL and 100 ng per mL of IGF-1, between 10 ng per mL and 20 ng per mL of FGF-4, between 100 ng per mL and 200 ng per mL of IL-6, between 5 ng per mL and 200 ng per mL of VEGF-A, and 40 nM of α-thrombin.
In another aspect, this document features a method for obtaining cells having the ability to differentiate into cardiomyocytes, wherein the method comprises contacting stem cells with a composition under conditions wherein the stem cells differentiate into the cells, wherein the composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that the composition comprises BMP, α-thrombin, or TNF-α when the composition comprises less than six of the molecules of the group. The cells can comprise Nkx2.5 polypeptides associated with the nuclei of the cells and MEF2C polypeptides associated with the nuclei of the cells. The cells can comprise Nkx2.5 polypeptides associated with the nuclei of the cells, MEF2C polypeptides associated with the nuclei of the cells, and GATA4 polypeptides associated with the nuclei of the cells. The cells can maintain the ability to differentiate into cardiomyocytes for 10 cell divisions if the cells are contacted with the composition for two days and the composition is removed from the cells after two days. The cells can maintain the ability to differentiate into cardiomyocytes for 10 cell divisions if the cells are contacted with the composition for five days and the composition is removed from the cells after five days. The cells can form a sarcomere if the cells are contacted with the composition for 15 days. The cells can produce a calcium transient in response to an electrical current if the cells are contacted with the composition for 21 days. The cardiomyocytes can be human cardiomyocytes. The stem cells can express CD105, CD166, CD29, and CD44 polypeptides, and can lack expression of CD14, CD34, and CD45 polypeptides. The stem cells can be mesenchymal stem cells. The stem cells can be human mesenchymal stem cells. The stem cells can be obtained from human bone marrow. The at least one of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. Each of the TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin can be a human polypeptide. The composition can comprise between 2.5 ng per mL and 10 ng per mL of the TGF-β. The composition can comprise between 5 ng per mL and 20 ng per mL of the BMP. The composition can comprise between 5 ng per mL and 50 ng per mL of the TNF-α. The composition can comprise between 1×10−6 μM and 2×10−6 μM of the retinoic acid. The composition can comprise between 50 ng per mL and 100 ng per mL of IGF-1, between 10 ng per mL and 20 ng per mL of FGF-4, between 100 ng per mL and 200 ng per mL of IL-6, between 5 ng per mL and 200 ng per mL of VEGF-A, and 40 nM of α-thrombin. The composition can comprise BMP, α-thrombin, and TNF-α when the composition comprises less than six of the molecules of the group.
In another aspect, this document features a method for providing heart tissue with cardiomyocytes, wherein the method comprises administering, to the heart tissue, cells obtained by contacting stem cells with a composition, wherein the composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.



DESCRIPTION OF THE DRAWINGS
FIG. 1A is a photomicrograph of untreated human mesenchymal stem cells immunostained with Nkx2.5 and α-actinin antibodies. FIG. 1B is a photomicrograph of TGF-β-treated human mesenchymal stem cells immunostained with Nkx2.5 and α-actinin antibodies. FIG. 1C is a photomicrograph of BMP-treated human mesenchymal stem cells immunostained with Nkx2.5 and α-actinin antibodies. FIG. 1D is a photomicrograph of a section of heart tissue from a wild-type mouse. FIG. 1E is a photomicrograph of a section of heart tissue from a transgenic mouse overexpressing a TNF-α polypeptide under the control of a cardiac-specific promoter. FIG. 1F is a photograph of a Western blot detecting TGF-β polypeptide expression in heart extracts from wild-type mice (WT) and transgenic mice overexpressing a TNF-α polypeptide under the control of a cardiac-specific promoter (TG). FIG. 1G is a photomicrograph of fluorescent stem cells engrafted into the myocardium of a transgenic mouse overexpressing a TNF-α polypeptide under the control of a cardiac-specific promoter.
FIG. 2A is a genomic fingerprint of cardiogenic endodermal cells that were or that were not stimulated with a TNF-α polypeptide. FIG. 2B is a table listing the cardiogenic outcome of human mesenchymal stem cells treated with the indicated combinations of cardiogenic factors. FIG. 2C is a photomicrograph of a human mesenchymal stem cell treated with regimen 9 of FIG. 2B and stained with DAPI and antibodies to Nkx2.5 and α-actinin.
FIG. 3 contains a series of photomicrographs of human mesenchymal stem cells that were stimulated for 2 days (panels a and b), 5 days (panels c and d), 15 days (panels e and f), or 21 days (panel g) with regimen 9 of FIG. 2B and stained with antibodies to Nkx2.5 (panels a, c, and e) or MEF2C (panels b, d, and f) as well as antibodies to α-actinin (panels a-f). Panel g is a photomicrograph of a cell loaded with Fluo 4-AM to monitor rhythmic calcium transient activity upon pacing (1 Hz).



DETAILED DESCRIPTION
This document provides methods and materials related to cardiac cells and cells capable of differentiating into cardiac cells. For example, this document provides cells having the ability to differentiate into cardiac cells (e.g., cardiomyocytes), cardiac cells obtained from such cells, methods for making such cells, compositions for making such cells, and methods for using such cells to provide heart tissue with cardiac cells.
Cardiac cells can be any type of heart cells. For example, cardiac cells can be mammalian (e.g., human) heart cells. In some cases, cardiac cells can be cardiomyocytes. Cells having the ability to differentiate into cardiac cells can be any type of cells having the ability to differentiate into cardiac cells. For example, cells having the ability to differentiate into cardiac cells can be mammalian (e.g., human) cells having the ability to differentiate into cardiac cells. In some cases, cells having the ability to differentiate into cardiac cells can be referred to as cardiopoietic cells. The term cardiopoietic cell used herein refers to a cell having the ability to differentiate into a cardiomyocyte.
A cardiopoietic cell can be associated with a cardiac transcription factor. For example, a cardiopoietic cell can have a Nkx2.5, a MEF2C, or a GATA4 polypeptide, or any combination thereof associated with its nucleus. For example, a cardiopoietic cell can have a Nkx2.5, a MEF2C, and a GATA4 polypeptide associated with its nucleus. In some cases, the cardiopoietic cell can have a Nkx2.5, a MEF2C, or a GATA4 polypeptide, or any combination thereof associated with its cytoplasm. In some cases, a cardiopoietic cell can have one or more of a Nkx2.5, a MEF2C, or a GATA4 polypeptide associated with its nucleus and one or more of a Nkx2.5, a MEF2C, or a GATA4 polypeptide associated with its cytoplasm. For example, a cardiopoietic cell can have a Nkx2.5 polypeptide associated with its nucleus and a MEF2C polypeptide associated with its cytoplasm.
Any method can be used to obtain the cardiopoietic cells. For example, the cardiopoietic cells can be derived from stem cells such as mammalian (e.g., human) stem cells. In some cases, the cardiopoietic cells can be derived from embryonic stem cells. In one embodiment, the cardiopoietic cells can be derived from mesenchymal stem cells. Mesenchymal stem cells can be obtained from any source. For example, mesenchymal stem cells can be obtained from mammalian (e.g., human) tissue such as bone marrow and trabecular bone. Mesenchymal stem cells can be cultured in vitro. For example, mesenchymal stem cells can be expanded in number in vitro. The mesenchymal stem cell can express or not express a polypeptide marker on its cell surface. For example, the mesenchymal stem cell can express CD105, CD16, CD29, and CD44 on its cell surface and not express CD14, CD34, and CD45 on its cell surface.
Any method can be used to derive cardiopoietic cells from stem cells (e.g., mesenchymal stem cells). For example, cardiopoietic cells can be derived from mesenchymal stem cells by incubating the mesenchymal stem cells with a composition. The composition can be any composition containing one or more factors. The factors can be any type of factors such as polypeptides, steroids, hormones, and small molecules. Examples of such factors include, without limitation, TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin. TGF-β can be any polypeptide having TGF-β activity, such as human TGF-β. For example, TGF-β can be recombinant TGF-β or synthetic TGF-β. In one embodiment, TGF-β can be TGF-β1. Any concentration of TGF-β can be used. For example, between 2.5 and 10 ng per mL of TGF-β can be used. BMP can be any polypeptide having BMP activity, such as human BMP. For example, BMP can be recombinant BMP or synthetic BMP. In one embodiment, BMP can be BMP-2. Any concentration of BMP can be used. For example, between 5 and 20 ng per mL of BMP can be used. TNF-α can be any polypeptide having TNF-α activity, such as human TNF-α. For example, TNF-α can be recombinant TNF-α or synthetic TNF-α. Any concentration of TNF-α can be used. For example, between 5 and 50 ng per mL of TNF-α can be used. IGF-1 can be any polypeptide having IGF-1 activity, such as human IGF-1. For example, IGF-1 can be recombinant IGF-1 or synthetic IGF-1. Any concentration of IGF-1 can be used. For example, between 50 ng per mL and 100 ng per mL of IGF-1 can be used. FGF-4 can be any polypeptide having FGF-4 activity, such as human FGF-4. For example, FGF-4 can be recombinant FGF-4 or synthetic FGF-4. Any concentration of FGF-4 can be used. For example, between 10 ng per mL and 20 ng per mL of FGF-4 can be used. IL-6 can be any polypeptide having IL-6 activity, such as human IL-6. For example, IL-6 can be recombinant IL-6 or synthetic IL-6. Any concentration of IL-6 can be used. For example, between 100 ng per mL and 200 ng per mL of IL-6 can be used. LIF can be any polypeptide having LIF activity, such as human LIF. For example, LIF can be recombinant LIF or synthetic LIF. Any concentration of LIF can be used. For example, between 2.5 ng per mL and 100 ng per mL of LIF can be used. VEGF-A can be any polypeptide having VEGF-A activity, such as human VEGF-A. For example, VEGF-A can be recombinant VEGF-A or synthetic VEGF-A. Any concentration of VEGF-A can be used. For example, between 5 ng per mL and 200 ng per mL of VEGF-A can be used. Retinoic acid can be any molecule having retinoic acid activity, such as synthetic retinoic acid, natural retinoic acid, a vitamin A metabolite, a natural derivative of vitamin A, or a synthetic derivative of vitamin A. Any concentration of retinoic acid can be used. For example, between 1×10−6 and 2×10−6 μM of retinoic acid can be used. α-Thrombin can be any polypeptide having α-thrombin activity, such as human α-thrombin. For example, α-thrombin can be recombinant α-thrombin or synthetic α-thrombin. Any concentration of α-thrombin can be used. For example, between 20 nM and 80 nM (e.g., 30 nM, 35 nM, 40 nM, 45 nM, or 50 nM) of α-thrombin can be used.
A composition provided herein can contain any combination of factors. For example, a composition provided herein can contain TGF-3, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin. In some cases, a composition provided herein can contain TGF-β, BMP, IGF-1, FGF-4, IL-6, LIF, retinoic acid, and α-thrombin. In some cases, a composition provided herein can contain TGF-β, BMP, IGF-1, FGF-4, IL-6, LIF, and VEGF-A. In some cases, a composition provided herein can contain BMP, IGF-1, FGF-4, IL-6, and LIF. In some cases, a composition provided herein can contain TGF-β, BMP, IGF-1, FGF-4, and α-thrombin. In some cases, a composition provided herein can contain TGF-β, BMP, TNF-α, IGF-1, and α-thrombin. In some cases, a composition provided herein can contain TGF-β, BMP, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin. In some cases, a composition provided herein can contain TGF-β, BMP, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin.
A composition provided herein can be prepared using any method. For example, a composition provided herein can be prepared using commercially available factors. In some cases, a composition provided herein can be prepared using conditioned medium from cells such as cardiomyocyte cells or TNF-α-stimulated endodermal cells. In some cases, a composition provided herein can be prepared using conditioned medium supplemented with commercially available factors. In some cases, a composition provided herein can be prepared using factors isolated from conditioned medium. In some cases, the factors can be dissolved in medium such as cell culture medium that does or does not contain serum.
Any method can be used to incubate stem cells (e.g., mesenchymal stem cells) with a composition provided herein. For example, mesenchymal stem cells can be incubated with a composition provided herein for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days. In some cases, a composition provided herein and used to incubate the mesenchymal stem cells can be replaced everyday or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days. In some cases, mesenchymal stem cells can be incubated with a composition provided herein in the presence or absence of serum. In some cases, mesenchymal stem cells can be incubated with a composition provided herein in vitro or in vivo.
Once the mesenchymal stem cells have been incubated with a composition provided herein, differentiation of the mesenchymal stem cells can be monitored to determine whether or not the mesenchymal stem cells have differentiated into cardiac cells. For example, the cells can be tested for expression of a cardiac transcription factor such as Nkx2.5, MEF2C, GATA4, or any combination thereof. Any method can be used to test the cells for expression of a cardiac transcription factor including Western blotting, fluorescence-activated cell sorting (FACS), immunostaining, and laser confocal microscopy. In some cases, incubation of mesenchymal stem cells with a composition provided herein for two days can result in nuclear translocation of Nkx2.5 and up-regulation of cytosolic MEF2C expression. In some cases, incubation of mesenchymal stem cells with a composition provided herein for five days can result in nuclear translocation of both Nkx2.5 and MEF2C. Differentiation of the mesenchymal stem cells can also be monitored by testing the cells for sarcomere formation. Any method can be used to test the cells for sarcomere formation including immunostaining using α-actinin antibodies and laser confocal microscopy. In some cases, incubation of mesenchymal stem cells with a composition provided herein for 15 days can result in sarcomere formation. In addition, differentiation of the mesenchymal stem cells can be monitored by testing the cells for functional excitation-contraction coupling. Any method can be used to test the cells for functional excitation-contraction coupling. For example, excitation-contraction coupling can be recorded using laser confocal line scanning in Fluo 4-AM loaded cells to assess intracellular calcium dynamics following electrical stimulation at 1 Hz, and Zeiss LSM Image software can be used to analyze the data. In some cases, incubation of mesenchymal stem cells with a composition provided herein for 21 days can result in functional excitation-contraction coupling with rhythmic calcium transient activity.
Any method can be used to provide heart tissue with cardiac cells. For example, cardiac cells can be injected into the coronary artery, infused in the heart, administered systemically, or injected transendocardially. Any heart tissue can be provided with cardiac cells. For example, mammalian (e.g., human) heart tissue can be provided with cardiac cells. In some cases, heart tissue that has suffered from ischemic cardiomyopathy, myocardial infarction, or heart failure can be provided with cardiac cells. Any type of cardiac cells can be administered to heart tissue. For example, autologous or heterologous cardiac cells can be administered to heart tissue. In some cases, stem cells (e.g., mesenchymal stem cells) that were incubated with a composition provided herein can be administered to heart tissue. The stem cells can be incubated with a composition provided herein for any length of time before being administered to heart tissue. For example, the stem cells can be incubated with a composition provided herein for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days before being administered to heart tissue. In some cases, stem cells that were incubated with a composition provided herein can be administered to heart tissue together with a composition provided herein. The stem cells can be incubated with a composition provided herein for any length of time before being administered to heart tissue together with a composition provided herein. For example, the stem cells can be incubated with a composition provided herein for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days before being administered to heart tissue together with a composition provided herein. In some cases, stem cells can be administered to heart tissue together with a composition provided herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1—Materials and Methods
Gene expression profiles of unprimed endodermal cells and endodermal cells primed with TNF-α were obtained by hybridizing labeled complementary RNA to the Mouse Genome 430 2.0 Array using standard protocols (Affymetrix, Santa Clara, Calif.). Data were acquired using a GeneChip Scanner 3000 (Affymetrix) and analyzed using GeneSpring software (Agilent Technologies, Palo Alto, Calif.). Data population sets were normalized to the unprimed or undifferentiated phenotype and quality filtered to eliminate background noise prior to hierarchical clustering. See, Behfar and Terzic, Nat Clin Pract Cardiovasc Med, 3 Suppl 1:S78-S82 (2006).
Mesenchymal stem cells were derived from human bone marrow withdrawn from the posterior iliac crest of the pelvic bone of 18- to 45-year-old healthy individuals (Cambrex, East Rutherford, N.J.). Based on flow cytometry analysis, the mesenchymal stem cells expressed CD105, CD166, CD29, and CD44, and did not express CD14, CD34, and CD45. The mesenchymal stem cells were cultured in DMEM (high glucose) containing 20% fetal bovine serum, penicillin, streptomycin, and L-glutamax (Invitrogen, Carlsbad, Calif.).
Human mesenchymal stem cells were plated at a density of 25,000 cells/25 cm2 Falcon flask (BD Biosciences, Bedford, Mass.). The cells were treated with one or more recombinant cardiogenic agents (Sigma, Saint Louis, Mo.) for up to 21 days. Cardiogenic transformation was monitored by laser confocal microscopy (Zeiss, Oberkochen, Germany) following immunostaining using MEF2C (1:400; Cell Signaling Technology, Beverly, Mass.), Nkx2.5 (1:300; Santa Cruz Biotechnology, Santa Cruz, Calif.), and α-actinin (1:1,000; Sigma) antibodies.
Excitation-contraction coupling was monitored using laser confocal line scanning in Fluo 4-AM (Invitrogen) loaded cells to assess intracellular calcium dynamics following electrical stimulation at 1 Hz. Zeiss LSM Image software was used to analyze the data.
Example 2—Stimulation of Mesenchymal Stem Cells with TGF-β or BMP
Human bone marrow-derived mesenchymal stem cells (Pittenger and Martin, Circ Res, 95:9-20 (2004)) were stimulated with TGF-β or BMP (FIG. 1A-C). FIG. 1A shows light green and red fluorescent staining surrounding the nucleus of the cells indicating the presence of Nkx2.5 and MEF2C, respectively. FIGS. 1B and 1C display brighter green and red fluorescent staining for both Nkz2.5 and MEF2C; in both figures, the green fluorescent staining is more pronounced in the nucleus of the cells, while the cytoplasm displays a higher concentration of the red fluorescent stain. In contrast to unstimulated mesenchymnal stem cells that had low expression levels of cardiac transcription factors (Nkx2.5 and MEF2C; FIG. 1A), stimulation with TGF-β or BMP up-regulated cytosolic expression of cardiac transcription factors (FIGS. 1B and 1C). Stimulation with TGF-β or BMP did not, however, promote nuclear translocation of cardiac transcription factors (FIGS. 1B and 1C). The increase in cytosolic expression of Nkx2.5 and MEF2C in response to stimulation with TGF-β or BMP indicated that the human mesenchymal stem cells had a cardiogenic potential. Induction with individual cardiogenic factors was sub-optimal, however, in that it did not promote nuclear translocation of cardiac transcription factors, which is required for cardiogenesis.
Example 3—Identification of Cardiogenic Factors
To advance the cardiac commitment of human bone marrow-derived mesenchymal stem cells, the factors necessary for cardiogenesis were identified. Cardiac-restricted transgenic overexpression of the cytokine TNF-α, which induces cardiomyopathy (Hodgson et al., EMBO J, 22:1732-1742 (2003); FIGS. 1D and 1E), was observed to stimulate TGF-β expression and result in cardiomyogenic transformation of transplanted stem cells (FIGS. 1F and 1G). FIG. 1G shows intense fluorescent blue staining indicative of overexpression of a TNF-α polypeptide. TGF-β alone does not induce cardiogenesis of human mesenchymal stem cells. Therefore, the effect of TNF-α was evaluated further in order to generate a comprehensive list of potential stem cell cardiogenic factors. A gene expression profile of cardiogenic endodermal cells (Mummery et al., Circulation, 107:2733-2740 (2003)) stimulated with TNF-α was generated using microarray technology. The mRNA levels of candidate cardiogenic factors were up-regulated (FIG. 2A). FIG. 2A displays an array of red and green strips with the green strips concentrated in the top two-thirds of the array, with an increase in red strips in the lower one-third. This list of candidate cardiogenic factors was refined by comparing it to the receptor profile of human mesenchymal stem cells (Pittenger and Martin, Circ Res, 95:9-20 (2004)) and selecting those factors for which the corresponding receptors are expressed on human mesenchymal stem cells. This list was further reduced to factors that induced up-regulation of cardiac transcription factors when applied to human mesenchymal stem cells. In addition to TGF-β, BMP, and TNF-α, these factors included insulin-like growth factor (IGF-1), fibroblast growth factor (FGF-4), interleukin 6 (IL-6), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF-A), retinoic acid (RA), and α-thrombin (FIG. 2B). A combination of at least five of the identified factors appeared necessary to induce a definitive cardiogenic response associated with nuclear translocation of cardiac transcription factors Nkx2.5 and MEF2C (FIGS. 2B and 2C). FIG. 2B displays an array similar to that seen in FIG. 2A, but the concentration of red and green striping is reversed. FIG. 2C illustrates a cell treated as in FIG. 2B, and shows a concentration of green (Nkx2.5) and blue (DAPI) staining in the nucleus with a large concentration of red (MEF2C) and a minor concentration of green staining in the surrounding area of the cell.
Example 4—Large-Scale Transformation of Mesenchymal Stem Cells Into Cardiopoietic Cells
Regimen 9 (FIG. 2B) was used for large-scale transformation of human bone marrow-derived mesenchymal stem cells into cardiac progenitors, ensuring their cardiogenic homogeneity for clinical applications. Human bone marrow-derived mesenchymal stem cells treated with the identified cardiogenic cocktail (regimen 9, FIG. 2B) exhibited, by day two of stimulation, consistent nuclear translocation of the early cardiac transcription factor Nkx2.5 (FIG. 3A) and cytosolic up-regulation of MEF2C, a later factor in cardiac differentiation (FIG. 3B). FIGS. 3A and 3B show bright blue staining in the nucleus of the cells surrounded by a majority of green and a minority of red staining in the surrounding area. By day five, stimulation with the cardiogenic cocktail induced nuclear migration of both Nkx2.5 and MEF2C (FIGS. 3C and 3D). FIGS. 3C and 3D display cells having a mixture of bright green and blue staining in the nucleus with additional green staining surrounding the nucleus. This phenotype was consistent with that of a cardioprogenitor cardiopoietic cell, an intermediate cell type distinct from the human mesenchymal stem cell source and committed to cardiac transdifferentiation. Indeed, sarcomere formation was evident by day 15 of stimulation with the cardiogenic cocktail (FIGS. 3E and 3F). FIGS. 3E and 3F display bright green and blue staining in the nucleus with only red staining observed in the surrounding cytoplasm. By day 21, functional excitation-contraction coupling with rhythmic calcium transient activity was recorded (FIG. 3G), indicating derivation of functional cardiac progeny. FIG. 3G shows an array of bright green fluorescent striping accented with further green staining in the intervening areas.
Other Embodiments
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. A method for providing heart tissue with cardiopoietic cells capable of differentiating into cardiomyocytes, wherein said method comprises:
(a) differentiating adult mesenchymal stem cells lacking expression of CD34 polypeptides into said cardiopoietic cells in vitro by contacting said adult mesenchymal stem cells with a composition under conditions wherein said adult mesenchymal stem cells differentiate into said cardiopoietic cells, wherein said composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that said composition comprises BMP, α-thrombin, or TNF-α when said composition comprises less than six of said molecules of said group; and
(b) administering said cardiopoietic cells to said heart tissue, wherein said cardiopoietic cells lack sarcomere formation as evidenced by an absence of a striated immunostaining pattern when stained with an anti-actinin antibody, and wherein said cardiopoietic cells have nuclear localization of Nkx2.5 polypeptides and MEF2C polypeptides.

2. The method of claim 1, wherein said cardiopoietic cells have nuclear localization of GATA4 polypeptides.

3. The method of claim 1, wherein said cardiopoietic cells are human cardiopoietic cells.

4. The method of claim 1, wherein said adult mesenchymal stem cells express CD105, CD166, CD29, and CD44 polypeptides and do not express CD14 and CD45 polypeptides.

5. The method of claim 1, wherein said adult mesenchymal stem cells are human mesenchymal stem cells.

6. The method of claim 1, wherein said adult mesenchymal stem cells are isolated from human bone marrow.

7. The method of claim 1, wherein at least one of said TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin is a human polypeptide.

8. The method of claim 1, wherein each of said TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, and α-thrombin is a human polypeptide.

9. A method for obtaining cardiopoietic cells having the ability to differentiate into cardiomyocytes, wherein said method comprises contacting adult mesenchymal stem cells lacking expression of CD34 polypeptides with a composition under conditions wherein said adult mesenchymal stem cells differentiate into said cardiopoietic cells, wherein said composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, provided that said composition comprises BMP, α-thrombin, or TNF-α when said composition comprises less than six of said molecules of said group, wherein said cardiopoietic cells lack sarcomere formation as evidenced by an absence of a striated immunostaining pattern when stained with an anti-actinin antibody, and wherein said cardiopoietic cells have nuclear localization of Nkx2.5 polypeptides and MEF2C polypeptides.

10. The method of claim 9, wherein said cardiopoietic cells have nuclear localization of GATA4 polypeptides.

11. The method of claim 9, wherein said cardiopoietic cells maintain the ability to differentiate into cardiomyocytes for 10 cell divisions if said cardiopoietic cells are contacted with said composition for two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days and said composition is removed from said cardiopoietic cells after said two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days.

12. The method of claim 9, wherein said adult mesenchymal stem cells express CD105, CD166, CD29, and CD44 polypeptides and do not express CD14, CD34, and CD45 polypeptides.

13. The method of claim 9, wherein said adult mesenchymal stem cells are human mesenchymal stem cells.

14. The method of claim 9, wherein said composition comprises between 2.5 ng per mL and 10 ng per mL of said TGF-β.

15. The method of claim 9, wherein said composition comprises between 1×10−6 μM and 2×10−6 μM of said retinoic acid.

16. A method for providing heart tissue with cardiopoietic cells capable of differentiating into cardiomyocytes, wherein said method comprises administering said cardiopoietic cells to said heart tissue, wherein said cardiopoietic cells are obtained by contacting adult mesenchymal stem cells lacking expression of CD34 polypeptides with a composition, wherein said composition comprises at least five molecules selected from the group consisting of TGF-β, BMP, TNF-α, IGF-1, FGF-4, IL-6, LIF, VEGF-A, retinoic acid, and α-thrombin, wherein said cardiopoietic cells lack sarcomere formation as evidenced by an absence of a striated immunostaining pattern when stained with an anti-actinin antibody, and wherein said cardiopoietic cells have nuclear localization of Nkx2.5 polypeptides and MEF2C polypeptides.

17. The method of claim 1, wherein said mesenchymal stem cells are in contact with said composition for one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days.