WO2008081457A2

WO2008081457A2 - Methods of isolating cardiac stem cells, banking and uses thereof

Info

Publication number: WO2008081457A2
Application number: PCT/IL2008/000026
Authority: WO
Inventors: Ayelet Itzhaki-Alfia; Israel Barbash; Jonathan Leor
Original assignee: Ramot At Tel Aviv University Ltd.; Tel Hashomer Medical Research Infrastructure And Services Ltd.
Priority date: 2007-01-04
Filing date: 2008-01-03
Publication date: 2008-07-10
Also published as: WO2008081457A3

Abstract

A method of isolating cardiac stem cells is disclosed. The method comprises contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase II under conditions sufficient to induce cell dissociation. Banks of the isolated cardiac stem cells are also disclosed.

Description

METHODS OF ISOLATING CARDIAC STEM CELLS, BANKING AND USES THEREOF

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a method of isolating stem cells and more particularly, to a method of isolating stem cells from the heart and the epicardial and pericardial fat thereof.

Myocardial infarction (Ml) is a life-threatening event and may cause cardiac sudden death or heart failure. Despite considerable advances in the diagnosis and treatment of heart disease, cardiac dysfunction after Ml is still the major cardiovascular disorder that is increasing in incidence, prevalence, and overall mortality. Following acute myocardial infarction, damaged cardiomyocytes are gradually replaced by fibroid nonfunctional tissue. Ventricular remodeling results in scar thinning and loss of regional contractile function. The ventricular dysfunction is primarily due to a massive loss of cardiomyocytes.

Since adult cardiomyocytes have little regenerative capability, it is widely accepted that the loss of cardiac myocytes after Ml is irreversible. Each year more than half million Americans die of heart failure. The relative shortage of donor hearts forces researchers and clinicians to establish new approaches for treatment of cardiac dysfunction in Ml and heart failure patients.

Recently, cell transplantation has emerged as a potential novel approach for regeneration of damaged myocardium. There have been a few attempts to regenerate the necrotic tissue by transplanting cardiomyocytes or skeletal myoblasts (Leor et al., Circulation. 1996 Nov 1;94 (9 Suppl) II332-6; Pouzet et al., Circulation. 2000 Nov 7;102(19 Suppl 3):III21O- 5). While the cells may survive following transplantation, they fail to reconstitute healthy myocardium and coronary vessels that are both functionally and structurally sound. Stem cell therapy to restore infarcted myocardium has been extensively studied, with numerous reports that mesenchymal stem cells derived from bone marrow (BMCs) can give rise to new myocardium via transdifferentiation (Mangei et al., Nat. Med. 9:1195-201, 2003). This in turn has rapidly translated into a whirlwind of clinical activity aimed at duplicating these effects in the human heart [see e.g. Strauer et al., Circulation 106:1913-1918, 2002; Assmus et al., Circulation 106: 3009-3017, 2002].

However, recent studies have rigorously challenged the conclusions of these reports by independently demonstrating that BMCs transplanted into damaged hearts could not give rise to cardiomyocytes [Balsam et al., Nature 428: 668-73, 2004; Murry et al., Nature 428: 664-8, 2004; Nygren et al., Nat. Med. 10: 494-501 , 2004). It has been noted that the beneficial effects noted in earlier studies in terms of ventricular performance might be partially attributable to angioblast-mediated vasculogenesis (Kocher et al., Nat. Med. 7:430-6, 2001) which could prevent apoptosis of native cardiomyocytes rather than by direct myogenesis.

Given the limitations of BMCs, it has become increasingly necessary to search for naturally occurring, authentic cardiac stem or progenitor cells and to develop novel methods of isolating same. Recently stem cells from myocardial tissue (CSCs) have been reported to generate cells with endothelial or myogenic properties, raising expectations that they could be used to help the heart to repair itself, and restore cardiac function [Laugwitz et al., Nature 433 (2005) 647-653; Messina et al., Circ Res. 2004;95:911-921]. In addition to cell and tissue therapy, the ability to selectively produce differentiated cell types from CSCs would be of great clinical importance in investigating the effects of drugs and environmental factors on human cell function.

However, current published methods for isolation of cardiac stem cells are complicated, and irreproducible. For example, Messina et al [Circ Res. 2004;95:911-921] isolated a mixed cardiac cell population by dissociation with a cocktail of 0.2 % trypsin and 0.1 % collagenase IV. These cells showed a phenotype associated with stem cells, endothelial and myogenic lineage. Following longer culture, the only conserved stem cell marker was c- kit (CD117).

Laugwitz et al [Nature 433 (2005) 647-653] identified isl1* cardiac progenitor cells during histology studies of right atrial tissues from 5 newborns up to 8-days old, but not in 148- day old newborns. To date, this group did not report any successful isolation method for isolating this kind of cardiac progenitors from human myocardial tissue.

Thus, there remains a need for, and it would be highly advantageous to have simple methods of isolating high numbers of viable cardiac progenitor cells.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of isolating cardiac stem cells, the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation, thereby isolating the cardiac stem cells.

According to an aspect of some embodiments of the present invention there is provided a cell bank, comprising a plurality of viable cardiac stem cell samples, isolated according to the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation, wherein the samples are from a plurality of individuals; and a database containing one or more data fields that allow for specific identification and retrieval of individual samples.

According to an aspect of some embodiments of the present invention there is provided a business method, the method comprising: (a) isolating cardiac stem cells according to the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation; and (b) licensing the right to further develop and/or store the isolated cardiac stem cells to a third party.

According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a cardiac disorder, the method comprising providing to the subject a therapeutically effective amount of the cells isolated according to the method comprising: (a) isolating cardiac stem cells according to the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation, thereby treating the subject having a cardiac disorder.

According to an aspect of some embodiments of the present invention there is provided a method of isolating cardiac stem cells, the method comprising dispersing fat tissue of a heart, thereby isolating the cardiac stem cells.

According to an aspect of some embodiments of the present invention there is provided a cardiac stem cell isolated by dispersing fat tissue of a heart.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the cardiac stem cells isolated by dispersing fat tissue of a heart.

According to an aspect of some embodiments of the present invention there is provided a method of treating a subject having a cardiac disorder, the method comprising providing to the subject a therapeutically effective amount of the cells isolated by dispersing fat tissue of a heart, thereby treating the subject having a cardiac disorder.

According to an aspect of some embodiments of the present invention there is provided a business method, the method comprising: (a) isolating cardiac stem cells by dispersing fat tissue of a heart; and (b) licensing the right to further develop and/or store the isolated cardiac stem cells to a third party.

According to some embodiments of the invention, the method further comprises purifying the cardiac stem cells following the cell dissociation.

According to some embodiments of the invention, the cardiac stem cells are adult cardiac stem cells.

According to some embodiments of the invention, the cardiac stem cells are embryonic stem cells.

According to some embodiments of the invention, the yield of cardiac stem cells is greater than 1.5 million cells per gram of the cardiac tissue. According to some embodiments of the invention, the composition further comprises trypsin and DNAse.

According to some embodiments of the invention, a concentration of the dispase Il is in a range between about 2 and about 3 units per ml.

According to some embodiments of the invention, the concentration is about 2.4 units per ml.

According to some embodiments of the invention, the concentration of trypsin is in a range between 0.1 % and 0.5 %.

According to some embodiments of the invention, the concentration is about 0.25 %. According to some embodiments of the invention, the concentration of the DNAse is in a range between 0.05 mg per ml and 0.5 mg per ml. According to some embodiments of the invention, the concentration is about 0.121 mg per ml.

According to some embodiments of the invention, the conditions sufficient to induce cell dissociation comprise a temperature of 37 ⁰C. According to some embodiments of the invention, the duration of the contacting is from 7-15 minutes.

According to some embodiments of the invention, the purifying is effected by a fluorescent-activated cell sorter (FACS) or a magnetic-activated cell sorter (MACS).

According to some embodiments of the invention, the adult stem cells express CD117.

According to some embodiments of the invention, the method further comprises freezing the tissue prior to the contacting.

According to some embodiments of the invention, the method further comprises storing the tissue at 4 ⁰C prior to the contacting. According to some embodiments of the invention, the samples are cryopreserved.

According to some embodiments of the invention, the one or more data fields comprises tissue typing data.

According to some embodiments of the invention, the method further comprises marketing the isolated cardiac stem cells. According to some embodiments of the invention, the method further comprises distributing the isolated cardiac stem cells.

According to some embodiments of the invention, the fat tissue of the heart is derived from an epicardium or pericardium

According to some embodiments of the invention, the dispersing is effected by contacting the fat tissue of the heart with a composition comprising at least one enzyme selected from the group consisting of dispase, collagenase and trypsin.

According to some embodiments of the invention, the composition comprises dispase II.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a novel method for isolating cardiac stem cells.

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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a pie chart illustrating the source of the cardiac tissue samples. FIG. 2 is a bar graph illustrating the distribution of age of the patients from where the heart tissue was obtained. FIG. 3 is a pie chart illustrating the gender of the patients from where the heart tissue was obtained.

FIG. 4 is a bar graph illustrating the cardiac-related disorders of the patients from where the heart tissue was obtained.

FIG. 5 is a pie chart illustrating the illustrating the cardiac surgery underwent by the patients from where the heart tissue was obtained. (CABG = coronary artery bypass graft).

FIGs. 6A-B are microscopic views human cardiac cells isolated according to the method of the present invention at different time points. Figure 6A is a microscopic view of cultured cells, two days following digestion. Some of the cells attached to the plate (black arrow) and formed colonies and some floated in suspension (white arrows). Figure 6B is a microscopic view of cultured cells, 3 days following digestion. The cells created typical colonies (black arrow). There are still some viable cells floating in the suspension - indicating stem cell phenotype (white arrows).

FIG. 7 is a line graph illustrating the high proliferative capacity of the cells of the present invention. FIGs. 8A-F are microscopic view of cultured cells at different time points expressing stem cell and cardiac markers. Figure 8A: Cytospined cells, 3 days following isolation, show positive red staining (arrow) with anti human CD 133 (hematopoietic stem cell markers). Nuclei are stained positive with DAPI (blue nuclear staining). Figure 8B: Cytospined cells, 3 days following isolation, show green positive staining (arrows) with anti human MDR1 (multi drug transporter resistant form Pg-P super family). Figure 8C: Cytospined cells, 10 days following isolation, show red positive staining with anti human CD 133 (arrows). Figure 8D: Cytospined cells, 14 days following isolation, show positive staining with anti human CD31 (green, marker for young endothelial cells) and CD133 (arrow, orange). Figure 8E: Cytospined cells, 14 days following isolation, show green positive staining (arrow) with anti human GATA4 (early development transcriptional factor). Figure 8F: Cytospined cells, 14 days following isolation, show positive staining (arrow) with anti human MDR1 (green) and CD117 (c-kit) (red, stem cell factor).

FIGs. 9A-D are microscopic views of the injected cardiac stem cells into nude rat myocardium, 1 week following transplantation. Figure 9A: Injected cardiac stem cells into nude rat myocardium expressed human CD117 (red, mark with arrow) one week following injection. Nuclei are stained blue with DAPI. Figure 9B: Injected cardiac stem cells into nude rat myocardium expressed positive human CD133 (red, mark with arrow) one week following injection. Figure 9C: Higher magnificent demonstrates early sarcomere formation. Injected cardiac stem cells into nude rat myocardium developed into early cardiomyocytes and expressed typical striation and positive staining for human fetal cardiac α-actin (green, mark with arrow). Figure 9D: Injected cardiac stem cells into nude rat myocardium expressed human cardiac Troponin I (brown). Nuclei are stained blue with Hematoxylin (purple).

FIG. 10 are microscopic views of the injected cardiac stem cells into nude rat myocardium, 1 month following transplantation. Transplanted cells formed a stable graft expressing human cardiac markers such as human cardiac α-actin (green), and formed gap junction, (represented by positive staining for connexin 43-red) indicating that the implanted cells created a graft of human heart muscle and are connecting with each other. Nuclei are stained blue with DAPI.

FIGs. 11A-I are graphs illustrating the results of FACS analysis of cells isolated according to the method of the present invention. Figure 11 A shows that 22 % of the cardiac cell culture expresses the stem cell marker CD117 (stem cell factor receptor). Figure 11 B shows that 7 % of the cardiac cell culture expresses the stem cell marker IsM. Figure 11C shows that 60 % of the cardiac cell culture expresses the progenitor cell marker GATA4 (early development transcriptional factor). Figure 11D shows that 17.8 % of the cardiac cell culture expresses the proliferation marker Ki-67. Figure 11 E shows that 60 % of the cardiac cell culture expresses the cardiac marker α actin. Figure 11 F shows that less than 1 % of the cardiac cell culture expresses CD68, indicating that there are no macrophages in the culture. Figure 11G shows that less than 1 % the cardiac cell culture expresses Collagen, indicating that there are no fibroblasts in the culture. Figure 11H shows that less than 1 % of the cardiac cell culture expresses CD45, indicating that there are no hematopoietic cells in the culture. Figure 111 shows that less than 1 % of the cardiac cell culture expresses CD31, indicating that there are no endothelial cells in the culture.

FIG. 12 is a bar graph illustrating that the right atrium has the best yield of c-kit positive cells (as detected by FACS analysis). FIG. 13 is a bar graph illustrating that the right atrium has the best yield of IsM positive cells (as detected by FACS analysis).

FIGs. 14A-F are graphs illustrating the results of FACS analysis of epicardial fat cells isolated according to the method of the present invention. Figure 14A shows that 61 % of epicardial fat cells express the progenitor stem cell marker GATA 4 following 10 days in culture. Figure 14B shows that 17 % of epicardial fat cells express the progenitor stem cell marker GATA 4 following 1 month in culture. Figure 14C shows that 87 % of epicardial fat cells express the cardiac marker α actin following 10 days in culture. Figure 14D shows that 82 % of epicardial fat cells express the cardiac marker α actin following 1 month in culture. Figure 11E shows that less than 1 % of the epicardial fat cells express CD31, following 10 days in culture, indicating that there are no endothelial cells in the culture. Figure 11F shows that less than 1 % of the epicardial fat cells express CD31, following 1 month in culture, indicating that there are no endothelial cells in the culture.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a method of isolating cardiac stem cells. The present invention can be used to generate a bank of cardiac stem cells which may serve as an autologous pool of stem/progenitor cells for transplantation and heart repair without the risk of rejection or autoimmune reaction, and without the need for lifelong immunosuppressive therapy. The principles and operation of the isolation procedure according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Advances in stem cell therapy in general have promoted the use of stem cells as a viable option for restoring infarcted myocardium. Therefore, it has become increasingly necessary to search for naturally occurring, authentic cardiac stem (including progenitor cells) and to develop novel methods of isolating same. However, current published methods for isolation of cardiac stem cells are complicated, and irreproducible. Furthermore, no efficient method to isolate these cells from human myocardium is available. Whilst reducing the present invention to practice, the present inventors have uncovered that a novel cocktail of reagents comprising dispase, DNase and trypsin is effective at dissociating adult myocardial tissue such that a significant percentage of viable stem cells may be subsequently purified.

Using this unique cocktail, the present inventors were able to isolate cardiac cells from adult human atrial and ventricular tissue where approximately 80 % of the non-myocyte cells remained viable following cell extraction. Unlike other methods used to isolate stem cells residing in the heart, the method of the present invention is reproducible and has a significant yield of cardiac stem cells from different zones within the human myocardium (Figure 1) and is not limited regarding age (up to 80 years) (Figure 2). The isolated cardiac stem cells showed a high proliferation capacity (Figure 7) and expressed typical stem cell markers (Figures 8A-F and Figures 11 A-I). When transplanted into athymic nude rat myocardium, a fraction of the cardiac stem cells isolated as described above developed into cardiomyocytes expressing human cardiac markers such as human cardiac α-actin (Figure 9C), and cardiac troponin I (Figure 9D). In addition, the transplanted cardiac stem cells isolated as described above formed gap junction, (represented by positive staining for connexin 43) indicating that the transplanted cells created a graft of human heart muscle and are connected with each other (Figure 10).

Furthermore, the cocktail described herein was used to isolate cardiac stem cells from the fat surrounding the heart tissue. The isolated cardiac stem cells expressed progenitor cell markers such as GATA 4 and cardiac markers such as cardiac α-actin

(Figures 14A-F). The present inventors thus deduced that cardiac fat tissue is a rich source of cardiac progenitor cells.

Thus, according to one aspect of the present invention there is provided a method of isolating cardiac stem cells, the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation.

As used herein, the term "isolating" refers to removing cardiac stem cells that from their naturally-occurring in-vivo environment (i.e. from cardiac tissue). Preferably the isolated cardiac stem cells of this aspect of the present invention are dissociated from other cells that are present in its in-vivo environment and even more preferably are dispersed into a single cell suspension.

As used herein, the term "tissue" refers to cardiac tissue of mammalian origin which comprises stem cells of interest. Preferably, the tissues are retrieved from humans. According to one embodiment, the tissue comprises cardiac fat tissue - e.g. pericardial and/or epicardial fat tissue. An exemplary location for cardiac fat tissue is covering the heart surface, jailed between the epicardium and the pericardium. Tissues of the present invention may be derived from subjects of any age (e.g., from embryos, fetuses, juveniles, and adults).

Any method may be used for retrieving the cardiac tissue sample from the mammalian subject - e.g. by surgery, during a biopsy, as long as it does not affect the viability or function of the stem cells within. The tissue may be pre-treated prior to isolation of the stem cells (e.g. cut or minced) in order to aid in the isolation of the stem cells therefrom. Preferably, the tissue is pre-treated in such a way that it does not negatively affect the viablility of the stem cells residing within (e.g., keeping the stem cells intact).

The tissue may be stored prior to isolation of the cardiac stem cells (e.g. at 4 ⁰C for about 18 hours). The present inventors have shown that an 18 hour incubation period at 4 ⁰C does not significantly effect the stem cell's viability (reduction of about 10 %). Alternatively, or additionally the tissue may be frozen prior to isolation of the cardiac stem cells. Methods of freezing tissues while retaining cell viability are known in the art (e.g. cryopreservation).

The phrase "cardiac stem cell", refers to cells residing in the heart which are not terminally differentiated but which can give rise to more differentiated daughter cells (e.g., terminally differentiated daughter cells). The cardiac stem cells may be adult stem cells (e.g. expressing markers such as C-kit (CD117), IsM , Ki-67, GATA4, CD133, and OCT3 or 4) or embryonic stem cells.

The cardiac stem cells are isolated by contacting tissues in which they are residing with an enzyme capable of dispersing the cells of the tissue. Such enzymes include for example dispase, collagenase and trypsin.

As used herein the term "dispase II" refers to the amino-endo peptidase metalloenzyme produced by Bacillus polymyxa (EC 3.4.24.4). Dispase Il is widely commercially available e.g. from Roche Applied Science (Cat. No. 10165859001). Preferably, dispase Il is used at a concentration between 2 and 3 units per ml. According to a particularly preferred embodiment of this aspect of the present invention, the concentration of dispase Il is about 2.4 U/ml.

According to this aspect of the present invention, the tissue is contacted with the composition at temperatures and for a length of time sufficient to allow the cardiac stem cells to dissociate from the tissue, whilst not affecting stem cell viability and function. Exemplary conditions include about 7-15 minutes at 37 ⁰C.

According to a preferred embodiment of this aspect of the present invention, the dissociating composition comprises trypsin (e.g., EC 3.4.21.4) at a concentration range between about 0.1 % and 0.5 %, preferably at about 0.25 %). Trypsin is widely available from such Companies as Sigma Aldrich, Catalogue number T9935.

The dissociating composition may also comprise DNAse (e.g. EC 3.1.21.1) at a concentration range between about 0.05 mg per ml and 0.5 mg per ml. A particularly preferred concentration of DNAse is about 0.121 mg per ml. DNase is widely available from such companies as Sigma Aldrich, Catalogue number AMPD1. As mentioned, the dissociating composition may also comprise collagenase (e.g., E C

3.4.24.3). Typically collagenase is used as a 0.1 % solution.

The cardiac stem cells are typically dissociated in a container, e.g. a Petri dish or a flask. Following incubation, the supernatant may be collected and centrifuged e.g. at 1800 rcf

(500 g) for 10 minutes at 4 ⁰C so as to remove any non-dissociated cells. This procedure may be repeated a number of times so as to ensure that the retrieved end- population comprises mainly dissociated cells.

Cardiac stem cells isolated according to the teachings of the present invention may reach yields of about 1.5 million cells, more preferably 3 million cells, more preferably 4 million cells, more preferably 5 million cells, more preferably 6.5 million cells, more preferably 8.5 million cells, more preferably 10 million cells and even more preferably 11.5 million cells per gram of cardiac tissue.

Following the isolation procedure described above, the cardiac stem cells are typically dispersed amongst other cells (e.g. myocytes) which have also been dissociated from the tissue by the composition of the present invention. The cardiac stem cells may be further purified from such cells in order to obtain a more homogeneous population of cells. The cardiac stem cells can be purified from the other tissue cells following the isolation method of the present invention using a variety of the methods known to those of skill in the art such as microscopy, immunolabeling and fluorescence sorting, for example solid phase adsorption, FACS, MACS, and the like. In preferred embodiments, the cardiac stem cells are isolated through sorting, for example immunofluorescence sorting of certain cell- surface markers. Two methods of sorting well known to those of skill in the art are magnetic- affinity cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

Sorting techniques such as immunofluorescence-staining techniques involve the use of appropriate stem cell markers to separate cardiac stem cells from other cells in the culture. Appropriate cardiac stem cell markers that may be used to isolate adult stem cells from other tissue cells include but are not limited to MDR1, Oct 3, CD133, CD105 and CD117 (c-kit). According to a preferred embodiment of this aspect of the present invention, cardiac adult stem cells may be isolated by MACS or FACS through the use of a cell surface marker such as CD117. By this means, enriched populations of cell-surface marker positive adult stem cells may be obtained from the mixed population of tissue cells. Alternatively, the cells can be sorted to remove undesirable cells by selecting for cell-surface markers not found on the adult stem cells. In the case of cardiac adult stem cells isolated from the heart, the adult stem cells were found to be negative for the following cell-surface markers: CD31 and lectin.

The enriched cardiac stem cell populations obtained by sorting have at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 98 %, or 99 % cardiac stem cells.

It will be appreciated that the cardiac stem cells of the present invention may be expanded following the isolation procedure of the present invention. Additionally, or alternatively cardiac stem cells may be expanded following the optional purification steps described hereinabove. Many methods are known in the art for expanding stem cells. For example, cardiac stem cells may be expanded by seeding on plates coated by collagen- gelatin, and cultured in growth medium e.g. DMEM. The growth medium may comprise additional components which aid in maintaining the cells viable and/or enhance the rate of expansion. Examples of such additional components include serum (e.g. 10 % FCS), growth factors, antibiotics etc. Cardiac stem cells isolated according to the teachings of the present invention may be used in a transplant setting in the treatment (including prevention) of various conditions. As described in Example 3 of the Examples section hereinbelow, isolated cardiac stem cells were shown to be capable of developing into cardiomyocytes in vivo.

Thus, according to another aspect of the present invention, there is provided a method of treating a subject having a cardiac disorder. The method comprises providing to the subject the cells isolated according to the method of the present invention.

As used herein the term "treating" refers to preventing, alleviating or diminishing a symptom associated with a cardiac disease or disorder. Preferably, treating cures, e.g., substantially eliminates, the symptoms associated with the cardiac disorder. As used herein the term "subject" refers to any (e.g., mammalian) subject, preferably a human subject.

Examples of cardiac disorders include, but are not limited to myocardial infarction, ischaemic heart disease, conduction disorders, valvular heart diseases and heart failure The cardiac stem cells of the present invention may be administered per se or as part of a pharmaceutical composition. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients (i.e. cardiac stem cells) are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (cardiac stem cells) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1). The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

The isolated stem cells of the present invention may be frozen (e.g. in a medium comprising FBS+DMSO in -80 ⁰C over night and/or for longer periods of time in liquid nitrogen. The present inventors have shown that following thawing of liquid nitrogen frozen cardiac stem cells, approximately 75 % of the cells remained viable.

Thus, the present invention contemplates a bank (i.e. a physical collection) of cardiac stem cell samples which may be accessed when required. For long-term storage in the bank, typically the cardiac stem cells of the present invention are cryopreserved. The bank may comprise a single aliquot of a sample from a particular individual or more than one aliquot from the same sample. The cardiac stem cells may be generated according to the methods of the present invention.

According to a preferred embodiment of this aspect of the present invention, the bank comprises cells that have been tissue typed. This would enable retrieval of histocompatibly matched cells from the bank for a patient needing a transplant. Thus, prior to storage in the bank, preferably the cells are tested for their genotype and histocompatibility haplotype, as appropriate. Preferably, the genotyping is carried out at a resolution level that allows one of ordinary skill to determine the similarity between the adult stem cells and any intended recipient thereof. Genotyping can be carried out in a number of ways including but not limited to restriction fragment length polymorphism (RFLP).

The isolated cardiac stem cells can also be tested for their histocompatibility haplotype. A histocompatibility haplotype is a set of alleles at the histocompatibility gene loci that is used by the immune system to distinguish between self and non-self (i.e., foreign) tissues and/or cells. In humans, the major histocompatibility (MHC) locus is composed of four loci on the short arm of chromosome 6. Humans also have a set of minor histocompatibility loci. As an example, human leukocyte antigen (HLA) typing is commonly performed for various transplants such as hematopoietic cell transplants. Major and minor histocompatibility antigens are present on cell surfaces and are recognized by the immune system as an indicator of the origin of the cell or tissue. Cells or tissues that are viewed as foreign will usually be rejected by the recipient via a host versus graft immune response.

The bank also preferably comprises a database, preferably stored in one or more computer-readable media, which contains information for each stored sample.

The information record may comprise, in fields or subfields, information relating to identification and location of the stored adult stem cell line (including an identification code), date of storage of the sample, characteristics of the stored adult stem cells (including but not limited to origin, differentiative capacity, phenotype of the stored line according to for example stem cell markers, proliferation rate and/or doubling time of stored line, adult stem cell colony morphology including if available electronic images thereof), donor information, pathogen testing of the stored samples, tissue typing of the stored cells and the like. As used herein the term "about" refers to ? 10 %.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes l-lll Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes l-lll CeIHs₁ J. E., ed. (1994); "Current Protocols in Immunology" Volumes l-lll Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901 ,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281 ,521; Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Myocardial Tissue Processing and cell isolation MATERIALS AND METHODS Patient characteristics: Tissues samples were donated from patients undergoing open heart surgery or tissue samples from transplanted heart biopsies. Tissues samples were collected from 102 patients (66 % men 34 % women, age 0-80 years), undergoing open heart surgery or percutaneous biopsy.

Cell isolation: Cells were isolated from the tissues using enzymatic digestion. Tissues were cut into small pieces of 1 mm each, and were washed twice with PBS for 4 minutes. Tissue samples were incubated with a cocktail of trypsin 0.25 %, dispase Il (2.4 units per ml) and DNAse 0.121 mg per ml in 37 ⁰C for several minutes. Following incubation, the supernatant was collected and centrifuged at 1800 rcf (500 g) for 10 minutes at 4 ⁰C. This procedure was repeated 8 times. Cells were counted and seeded in 6 well plates coated by collagen-gelatin, at a concentration of 1*10⁴ cells per well and cultured in DMEM growth medium (containing 10 %

FCS).

Ce// isolation by Magnetic-Activated Cell Sorter (MACS): Following 5-10 days of culture, cardiac stem cells (CSC) were isolated using magnetic-activated cell sorter (MACS) based on antibodies against human CD117 (Miltenyi Biotec, Bergisch Gladbach, Germany).

CSC were labeled magnetically using magnetic antibody. Labeled cells were passed through a depletion column held within a magnetic apparatus (Miltenyi Biotec, Bergisch Gladbach,

Germany). As a result of the high-gradient magnetic field created within the depletion column matrix, labeled cells were retained within the magnetized column, while the negative cells passed freely through the magnetic field. After removal of the column from the magnetic field, the magnetically retained cells were eluted as the positively selected cell fraction. The fraction containing the positive cells was collected and washed with PBS.

RESULTS

The collected specimens were characterized by a few parameters (Figures 1-5). The specimens were obtained from the right atrium (47 %), left atrium (30 %), right ventricle (10 %), left ventrical (2 %), right septum (2 %), left septum (8 %) and apex (1 %).

The total number of cells in the extraction was related to the amount of tissue. In all cases, except left atrium, a small tissue sample (biopsy size 1-2 mm) was received. In the cases of tissue samples obtained from the left atrium, the patients underwent partial excision of the left atrium ("maze" operation). In these cases the tissue is significantly larger and therefore the total number of cells was higher.

Using magnetic-activated cell sorter based on antibodies against human CD117 (Miltenyi), the positive fraction contained ~ 22 % from total cell number.

The unique cocktail of the present invention had a very high reproducibility capacity. The present results demonstrated -100 % successes in cell extraction and cell viability from each tissues sample received.

The cocktail efficiency was also tested on biopsy tissues from patients undergoing catheterization. Cell number assembled in the extraction was related to the amount of tissue. In these cases the tissue is significantly smaller but, the total amount of cells were equal to isolation from other tissues (left ventricle, right ventricle, left atrium and right atrium).

The feasibility of storing the tissues at 4⁰C prior to cell isolation was also examined. Tissues that were kept in medium for 16 hours at 4 ° C, generated a similar number of cells to that retrieved from fresh tissues (reduction of 10 %).

EXAMPLE 2

Cardiac stem cell characterization lmmunofluorescent staining: Cells were removed from the plate by 0.05 % EDTA and re-suspended in PBS⁺BSA 0.1 % and cytospined (1^*10⁴ on each slide). Slides were fixed with methanol-acetone for 15 minutes. Incubation with primary antibodies MDR1 (Mouse IgG anti human, Chemicon), GATA-4 (Goat anti human, R&D), OCT3 (Rat anti human, R&D), CD133 (Mouse IgG anti human, Miltenyi), CD31 (Mouse IgG anti human, Dako), CD117 (Rabbit anti human, Dako) and cardiac alpha actin (Mouse IgG anti human, Acris) was performed for 30 minutes and the slides were washed twice in PBS for 15 minutes. Next, the slides were incubated with a secondary antibody (FITC, CY3, CY2) for 1 hour. Following incubation, the slides were washed twice in PBS for 15 min. Finally, the slides were incubated with DAPI (nuclear staining) for 15 minutes. RESULTS

The seeded cells following MACS isolation formed colonies. After a few days in culture, some viable cells were still floating in suspension, which indicated the presence of stem cells (Figures 6A-B).

Proliferation capacity of the isolated cells of the present invention: A significant proportion of the cells expressed a high proliferation capacity, cell number increased from 200,000 to 2,000,000 cells in 5 days of culture (doubling time =1.3 days as illustrated in Figure 7. Stem cell characteristics: To determine cell characteristics and the level of differentiation of the isolated cells, expression of several stem cell markers was analyzed. All immunostaining experiments were performed on cytospined cells, because the cells attach to plastic only and were not able to attach to cover slips or to coated cover slips. As illustrated in Figures 8A-F the cells stained positively with all the tested stem cell markers. Table 1 , herein below summarizes the temporal expression of stem cell and cardiac markers of the cells isolated according to the method of the present invention.

Table 1

EXAMPLE 3

Transplantation of the cardiac stem cells of the present invention MATERIALS AND METHOD

Cell transplantation: Athymic nude rats were anesthetized and under sterile technique the chest was opened. Rats were transplanted with either 2*10⁶ cells, or 150 μL of PBS, using a 27-gauge needle. RESULTS

To examine cell differentiation (in vivo) with host myocardium, cardiac stem cells isolated according to the method of the present invention were injected into athymic nude rat myocardium. In the first experiment, one week following the injection, the hearts were removed and processed for histological examination. Some cells still expressed stem cell markers but some cells expressed human cardiac markers such as human cardiac α-actin, with early sarcomere formation (Figure 9C), and cardiac troponin I (Figure 9D) - indicating that some of the implanted human cells developed into cardiomyocytes in vivo. In the second experiment, one month following injection, the hearts were removed and processed for histological examination. Transplanted cells formed a stable graft expressing human cardiac markers such as human cardiac α-actin, and formed gap junction, (represented by positive staining for connexin 43) indicating that the implanted cells created a graft of human heart muscle and are connecting with each other (Figure 10).

EXAMPLE 4

Further characterization of the cardiac stem cells of the present invention MATERIALS AND METHOD

Fluorescent-Activated Cell Sorter (FACS): To determine cell characteristics and differentiation levels, several markers of stem cell, progenitor cell and differentiated cell expressions were used (such as: endothelial, macrophage, hematopoietic and fibroblast).

Expanded cardiac cell characteristics were examined by a Fluorescent-Activated Cell Sorter

(FACS). Cardiac cells were labeled using the following primary antibodies.

CD117 - (stem cell factor receptor) (Rabbit anti human, Dako), lslet-1 (goat anti- human, Chemicon), GATA-4 - (early development transcriptional factor) (Goat anti-human, R&D), Ki-67 - (proliferation marker) Mouse IgG anti human, Dako), CD68 - (macrophages cells marker) (Mouse IgG anti human, Dako), CD45 - (hematopoietic cells marker) (Mouse IgG anti-human, Dako), CD31 - (endothelial cells marker) (Mouse IgG anti-human, Dako), CD117 (Rabbit anti-human, Dako), Collagen type 1 - (fibroblasts cells marker) (Mouse IgG anti human, Chemicon) and Cardiac alpha actin - (cardiac cells marker) (Mouse IgG anti- human, Acris).

Incubation with primary antibodies was performed for 30-45 minutes, after which cells were washed with PBS+0.1 %BSA (staining buffer). Next, the cells were incubated with a secondary antibody (FITC, CY3, CY2) for 1 hour, after which the cells were washed with 0.7-1 ml staining buffer and centrifuged for 10 minutes at 300 x g. Finally, cells were re-suspended in 0.5ml staining buffer for immediate analysis by FACS. RESULTS

Cardiac cell characteristics: The results are presented in Figures 11A-I. The results indicate that the cells isolated using the method of the present invention contain stem cells markers such as Cd117 (22 %), lsl-1(7 %), progenitor cells markers such as GATA 4 (60

%), and cardiac markers such as cardiac α-actin (60 %). In the culture there are less then

1 % of macrophages, fibroblast, hematopoetic and endothelial cells.

The best source of C-kit and IsI-I positive cells was shown to be from the right atrium (Figures 12 and 13).

EXAMPLE 5

Isolation of cardiac stem cells from epicardial fat tissues MATERIALS AND METHODS

Ce// isolation and culture: Isolation and culturing of cells from epicardial fat tissue was performed as described in Example 1.

Characterization of cells isolated from epicardial fat tissue: The collected specimens were examined by a Fluorescent-Activated Cell Sorter (FACS) to determine cell characteristics and differentiation levels.

Cardiac cells were labeled using primary antibodies: GATA-4 - (early development transcriptional factor; Goat anti-human, R&D), CD31 - (endothelial cells marker; Mouse IgG anti-human, Dako), Cardiac alpha actin - (cardiac cells marker; Mouse IgG anti- human,

Acris). Incubation with primary antibodies was performed for 30-45 minutes, following which, cells were washed with PBS+0.1%BSA (staining buffer). Next, the cells were incubated with a secondary antibody (FITC, CY3,) for 1 hour, after which the cells were washed with 0.7-1 ml staining buffer and centrifuged for 10 minutes at 300 x g. Finally, cells were re-suspended in 0.5ml staining buffer for immediate analysis by FACS. RESULTS

The results illustrated in Figures 14A-F indicate that cell cultures derived from epicardial fat contain progenitor cells markers such as GATA 4 (60 %) and cardiac α-actin (60 %). Less than 1 % of those cultures comprised endothelial cells.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of isolating cardiac stem cells, the method comprising contacting a tissue which comprises the cardiac stem cells with a composition which comprises dispase Il under conditions sufficient to induce cell dissociation, thereby isolating the cardiac stem cells.

2. The method of claim 1, further comprising purifying said cardiac stem cells following said cell dissociation.

3. The method of claim 1 , wherein said cardiac stem cells are adult cardiac stem cells.

4. The method of claim 1, wherein said cardiac stem cells are embryonic stem cells.

5. The method of claim 2, wherein a yield of cardiac stem cells is greater than 1.5 million cells per gram of said cardiac tissue.

6. The method of claim 1, wherein said composition further comprises trypsin and DNAse.

7. The method of claim 1 , wherein a concentration of said dispase Il is in a range between about 2 and about 3 units per ml.

8. The method of claim 7, wherein said concentration is about 2.4 units per ml.

9. The method of claim 6, wherein a concentration of said trypsin is in a range between 0.1 % and 0.5 %.

10. The method of claim 9, wherein said concentration is about 0.25 %.

11. The method of claim 6, wherein a concentration of said DNAse is in a range between 0.05 mg per ml and 0.5 mg per ml.

12. The method of claim 11, wherein said concentration is about 0.121 mg per ml.

13. The method of claim 1 , wherein said conditions sufficient to induce cell dissociation comprise a temperature of 37 ⁰C.

14. The method of claim 13, wherein a duration of said contacting is from 7-15 minutes.

15. The method of claim 2, wherein said purifying is effected by a fluorescent- activated cell sorter (FACS) or a magnetic-activated cell sorter (MACS).

16. The method of claim 3, wherein said adult stem cells express CD117.

17. The method of claim 1 , further comprising freezing said tissue prior to said contacting.

18. The method of claim 1, further comprising storing said tissue at 4 ⁰C prior to said contacting.

19. A cell bank, comprising a plurality of viable cardiac stem cell samples, isolated according to the method of claim 1, wherein said samples are from a plurality of individuals; and a database containing one or more data fields that allow for specific identification and retrieval of individual samples.

20. The cell bank of claim 19, wherein the samples are cryopreserved.

21. The cell bank of claim 19, wherein said one or more data fields comprises tissue typing data.

22. A business method, the method comprising:

(a) isolating cardiac stem cells according to the method of claim 1 ; and

(b) licensing the right to further develop and/or store said isolated cardiac stem cells to a third party.

23. The business method of claim 22, further comprising marketing said isolated cardiac stem cells.

24. The business method of claim 22, further comprising distributing said isolated cardiac stem cells.

25. A method of treating a subject having a cardiac disorder, the method comprising providing to the subject a therapeutically effective amount of the cells isolated according to the method of claim 1 , thereby treating the subject having a cardiac disorder.

26. A method of isolating cardiac stem cells, the method comprising dispersing fat tissue of a heart, thereby isolating the cardiac stem cells.

27. The method of claim 26, wherein said fat tissue of the heart is derived from an epicardium or pericardium

28. The method of claim 26, wherein said dispersing is effected by contacting said fat tissue of the heart with a composition comprising at least one enzyme selected from the group consisting of dispase, collagenase and trypsin.

29. The method of claim 26, wherein said composition comprises dispase II.

30. The method of claim 26, further comprising purifying said cardiac stem cells following said cell dissociation.

31. The method of claim 29, wherein said composition further comprises trypsin and DNAse.

32. The method of claim 29, wherein a concentration of said dispase Il is in a range between about 2 and about 3 units per ml.

33. The method of claim 32, wherein said concentration is about 2.4 units per ml.

34. The method of claim 30, wherein a concentration of said trypsin is in a range between 0.1 % and 0.5 %.

35. The method of claim 34, wherein said concentration is about 0.25 %.

36. The method of claim 31, wherein a concentration of said DNAse is in a range between 0.05 mg per ml and 0.5 mg per ml.

37. The method of claim 36, wherein said concentration is about 0.121 mg per ml.

38. The method of claim 28, wherein said contacting is effected at a temperature of 37 ⁰C.

39. The method of claim 38, wherein a duration of said contacting is from 7-12 minutes.

40. The method of claim 30, wherein said purifying is effected by a fluorescent- activated cell sorter (FACS) or a magnetic-activated cell sorter (MACS).

41. The method of claim 28, further comprising freezing said tissue prior to said contacting.

42. The method of claim 28, further comprising storing said tissue at 4 ⁰C prior to said contacting.

43. A cardiac stem cell isolated according to the method of claim 26.

44. A pharmaceutical composition comprising the cardiac stem cells of claim 43.

45. A method of treating a subject having a cardiac disorder, the method comprising providing to the subject a therapeutically effective amount of the cells isolated according to the method of claim 26, thereby treating the subject having a cardiac disorder.

46. A business method, the method comprising:

(a) isolating cardiac stem cells according to the method of claim 26; and