KR20070007030A - Novel multipotent stem cells and use thereof - Google Patents

Abstract

Disclosed is an isolated bone marrow stem cell (MBSC) having undetectable or low levels of selected cell markers including those typical of endothelial, neuronal and smooth muscle cells. Also disclosed are grafts pharmaceutical products that include such cells. Methods of making the BMSCs are also provided. The invention has a wide spectrum of useful applications including use in the prevention and treatment of cardiovascular disease. ® KIPO & WIPO 2007

Description

Novel multipotent stem cells and use thereof

The present invention generally relates to isolated bone marrow stem cells (BMSCs) having unsearchable or low levels of select cell markers. Importantly, the cells are pluripotent and can be used to make a variety of desirable cell types. The present invention has a wide range of useful applications, including use in the prevention and treatment of cardiovascular diseases.

There is increasing awareness that congestive heart failure (CHF) is an increasing global epithelial. Several causes suggested irreversible damage resulting from, for example, myocardial infraction (MI).

Infarct size is a major determinant of morbidity and mortality in CHF. For example, a large infarction affects more than 40% of the left ventricle (LV) and is associated with the rapid progression of cardiac shock or congestive heart disease, which is typically difficult to repair. The principle of many years is that myocardium has limited self-healing or regeneration ability, and also irreversible loss of muscles and concomitant contractions and fibrosis of myocardial wounds act as a series of events, progressive ventricular remodeling of nonischemic myocardium, which ultimately progresses. Induces HF

Efforts have been made to understand HF in more detail. For example, loss of cardiomyocyte (CMC) survival role is thought to be related to HF. This suggests some importance in design therapies that assist in maintaining viable cardiomyocytes during HF progression. Currently, clinically used drug treatments or procedures have shown efficacy in replacing myocardial wounds with functional contractile tissue. Thus, new approaches have been sought to explain the major pathophysiological deficiencies involved in these symptoms, such as muscle and CMC, in the case of major morbidity and mortality associated with MI and HF.

  Tissue specificity The belief that stem cells can only be distinguished as cells of the original tissue is widespread. However, it is emerging that at least certain adult tissue-specific stem cells can be distinguished by lineage other than the original tissue. This plasticity is referred to as trans-differentiation. Bone marrow cells appear to have the ability to regenerate many non-hematopoietic tissues, including neural ectoderm cells, skeletal stem cells, CMCs, endothelial, liver and biliary tract epithelium, and lung, digestive and dermal stem cells. Recently, a subset of BM derived stem cells referred to as pluripotent adult progenitor cells (MAPCs) has been purified with MSCs. MAPCs are thought to multiply extensively and differentiate into cells of all three layers.

Therapeutic application of BM derived stem cells has been attempted. Work has shown that CD34 (+) cells implanted into endothelial progenitor cells (EPCs), hemangioblasts, or ischemic myocardium are introduced at the center of neovascularization and also have a favorable impact on LV function after MI. The more diverse potential of BM derived hematopoietic stem cells (HSCs) has been demonstrated in other studies.

Another source of adult stem cells developed for possible cardiac regeneration is non-hematopoietic mesenchymal stem cells (or bone marrow stromal cells, MSCs). MSCs are derived from adult BM and may also have multiline differentiation capacity. MCSs in the medium can maintain stable phenotypes that have not been differentiated for many generations. Murine, bovine and human MSCs have been shown to be capable of myocardial differentiation in animal models of MI.

Although there are reports that SCs derived from BM have the potential to regenerate myocardial tissue, it is unclear whether these immature cells have many clinical potentials.

For example, while BM supports evidence that contains plastic populations with plasticity, isolation and expansion of non-differentiated SCs from single cell units is important as one way to increase treatment reliability and efficacy. Moreover, conventional attempts to culture some BM cells, such as MAPC, required the use of expensive cytokines to make them more attractive for clinical use. More importantly, MAPC showed minimal grafting into the myocardium after injection into blastocysts. Certain BM cells, such as Lin-c-KIT + cells or hemangioblasts, have been shown to reproduce a significant proportion of different myocardial tissues in vivo. However, these cells are very rare and lack a permitted culture.

There were other disadvantages associated with the use of BM, especially in therapeutic applications. For example, we do not demonstrate whether a single human SC participates in neovascular neoplasia and myogenicity in a myocardial wound model. EC differentiation has not been demonstrated in studies using human MSCs. Studies using MSCs have demonstrated differentiation into myocardial phenotype by immunohistochemical contamination. However, the number of cells introduced was small and the morphological characteristics were inconsistent with mature CMCs.

Therefore, these and other drawbacks are the limited use of BM as a cell source that can be used to prevent, treat or alleviate the severity of symptoms associated with heart disease such as myocardial infarction.

It is desirable to have BM cells that are convenient for isolation and maintenance in tissue culture as the clonal separates. It is particularly preferred if such BM cells can be maintained in pluripotent clones as a source of at least one and preferably all three of the EC, SMC and CMC cells in particular. It is more desirable if such BM cells can be transplanted into diseased or damaged cardiac tissue to prevent, treat or alleviate related symptoms.

We have developed a new class of pluripotent bone marrow stem cells (BMSCs) that can be cultured (expanded) from one or several bone marrow cells. Preferred BMSCs can proliferate for a long time and also have at least triple pass differentiation. The formation of these cells from BMSCs can be regulated and used to provide a potentially unlimited source of differentiated cells and tissues, including grafts. The present invention has a wide range of important applications, including treatment involving the migration of BMSCs (or cells derived therefrom) into prescriptions requiring treatment.

More specifically, the present invention provides bone marrow (BW) that can proliferate in medium without browsable or plastic loss for at least about 50 population doublings (PDs), more typically at least about 100 PDs, and typically about 140 PDs or more. New stem cell (SC) populations were isolated from. In one embodiment we expanded BMSCs (referred to as hBMSCs) from human bone marrow into clonals and they also contained certain known BM-derived SC groups such as hematopoietic SCs, mesenchymal SCs or pluripotent adult vulvar surface cells. For example, it does not belong to endothelial vulvar surface cells (EPCs). Importantly, preferred BMSCs of the present invention can differentiate into at least three different cell types when provided with appropriate signals, especially endothelial cells (ECs), flexible muscle cells (SMCs) and cardiomyocytes (CMCs). In this embodiment, the present invention is suitable for providing a potential infinite source of cells and tissues (including grafts) needed to prevent, treat or alleviate pain associated with a range of myocardial diseases including, for example, infarct related diseases.

Thus, and in one embodiment, the present invention provides the following cell markers at an unsearchable or low (negligible) degree as measured by standard cell marker search assays: CD90, CD117, CD34, CD113, FLK-1, tie-2 , Population of bone marrow stem cells (BMSCs) having at least one and preferably all of, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor To provide.

Such BMSCs have important uses and advantages. For example, BMSCs can be used to provide a potentially unlimited source of cells and tissues for treating diseases that are highly plastic and also exhibit cardiovascular disease, particularly regenerative treatment. Attempts have been made to use other SCs to treat these diseases, but these cells are relatively less plastic than the BMSCs described herein. Patients suffered from a lack of treatment that could provide a variety of cardiovascular cells and tissues. The present invention addresses this need by providing for the first time one or a variety of cardiovascular cells and in particular BMSCs that can be easily converted to ECs, SMCs and CMCs. In embodiments in which BMSCs are isolated from primates, such as human patients (hBMSCs), these cells may be ex vivo (eg, one or more mitogens) to maintain and / or treat and also to treat cardiovascular disease. It may be returned (transplanted) to the patient (or individual immunologically relevant patient, such as a family (member)). This feature of the invention helps, for example, to reduce the risk of unintentional infection of the virus and immunological rejection of the transplanted cells. Alternatively or additionally, BMSCs can be isolated from the patient and in particular embodiments can also be treated with dislodge (with or without mitogen) to produce a tissue implant that is at least allergic and preferably innate to the patient. . Such implants can be used to treat cardiovascular diseases derived therefrom, for example, which require a relatively large amount of BMSCs or cells.

The invention further provides a method for producing the isolated myeloid cells (eg, hBMSCs) described herein. In one embodiment the method comprises at least one and preferably all of the following steps.

a) collecting bone marrow cells from a mammal wherein the cells have a size of less than about 100 microns, preferably less than about 50 microns and more preferably about 40 microns or less,

b) culturing (expanding) the collecting cells in medium under conditions of selecting adherent cells,

c) selecting adherent cells and expanding these cells in semi-confluency in the medium,

d) diluting the cultured cells sequentially in a chamber having a controlled medium, wherein the dilution is dilution sufficient to form a density of less than about 1 cell per chamber that separates the expanded cells, and

e) incubating (expansion) each of the clonal isolates and then selecting a chamber with expanded cells to create an isolated bone marrow cell population.

In another embodiment, the present invention provides a tissue or implant comprising, but not limited to, isolated BMSCs described herein, such as hBMSCs.

In another aspect of the invention, a method of preventing, treating or alleviating the severity of a cardiovascular (heart) disease is provided. In one embodiment the method comprises administering at least one of the isolated BMSCs (eg, hBMSCs) described herein to a mammal in need thereof. Preferably, said administration is sufficient to prevent, treat or alleviate the severity of the disease in a mammal. Alternatively or additionally, the method comprises administering to the mammal at least one implant of the present invention under conditions that can aid in enhancing heart disease.

The present invention further provides pharmaceutical products that prevent, treat or alleviate the severity of heart disease. In one embodiment, the product comprises at least one of the following components: isolated BMSCs (eg, hBMSCs) and / or optionally separating cells from a mammal; Directions for making, maintaining and / or using the implant of the invention and / or optionally the implant; Cell cultures, tissues or organs of the present invention and / or optionally the directions for preparing them from a mammal.

The present invention also provides a population of isolated bone marrow cells obtained by at least one and preferably all of the following process steps.

a) collecting bone marrow cells from a mammal wherein the cells have a size of less than about 100 microns, preferably less than about 50 microns and more preferably about 40 microns or less,

b) culturing (expanding) the collecting cells in medium under conditions of selecting adherent cells,

c) selecting adherent cells and developing these cells in the semi-adhesion point in the medium.

d) diluting the cultured cells sequentially in a chamber having a controlled medium, wherein the dilution is dilution sufficient to form a density of less than about 1 cell per chamber that separates the expanded cells, and

e) incubating (expansion) each of the clonal isolates and then selecting a chamber with expanded cells to create an isolated bone marrow cell population.

  Other features, uses and advantages of the invention are described below.

1A-E show the characteristics of human bone marrow stem cells (hBMSC) of the present invention.

2A-D show ex vivo differentiation of hBMSCs in endothelial cell (EC) and flexible muscle cell (SMC) passages.

3A-M show ex vivo differentiation of hBMSc in neutral and endoderm passages.

4A-X show ex vivo trans-differentiation and fusion of hBMSCs to cardiomyocytes (CMC), EC and SMC cells.

5A-F show that hBMSC transplantation attenuates reversible heart remodeling in a rat model of myocardial infarction (MI).

6A-T show grafting and multiple passage differentiation of transplanted hBMSCs in infarct myocardium.

7A-U show that human BMSC transplantation enhances myocardial cell proliferation and reduces myocardial apoptosis.

8A-B show that hBMSC transplantation overregulates mRNA expression of angiogenic cytokines and cardiac transcription.

9A-L show that human BMSC transplantation increases capillary and CMC density and also increases myocardial fibrosis.

10 shows the results of FACS analysis using multiple surface epitopes.

As mentioned above, the present invention provides the following cell markers of unsearchable or low (negligible) degree: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5 , A population of bone marrow stem cells (BMSCs) having at least one and preferably all of Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor. Such markers can be readily determined as mentioned herein as standard cell marker search assays. The BMSCs of the present invention can be used for the prevention, treatment or Has a wide range of important uses, including use in mitigation.

The term “standard cell marker search assay” refers to a conventional immunological or molecular assay configured to search and optionally quantify one of the cell markers described above (ie CD90, CD117, CD34, etc.). Examples of such conventional immunological assays include western blotting, ELISA and RIA. Preferred antibodies used in such assays are described below. Generally Harlow and Lane in Antibodies : A Laboratory Manual , CSH Publications, NY (1988), describing these and other suitable assays. Particular molecular assays suitable for this use include polymerase chain reaction (PCR) type assays using oligonucleotide primers described herein. For a general description of recombinant PCR and related methods, see WO92 / 07075. Also, a general description of cognitive immunological and molecular assays that can be used to explore cellular markers is described in Sambrook et al. in Molecular Cloning : A Laboratory Manual (2d ed. 1989); And Ausubel et al., Current Protocols in Molecular See Biology , John Wiley & Sons, New York, 1989.

As used herein, the term “isolated” means that a cell is isolated from naturally occurring bone marrow and other cell substituents. Preferably, the BMSCs of the invention and in particular hBMSCs having at least 98 to 99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified or isolated, BMSCs are rarely free of unwanted bone marrow contaminants. Once partially purified or purified to substantially pure water, BMSCs are suitable for therapeutic use or other uses as described herein. Purity can be determined by various standard techniques such as cell culture, microscopic and centrifugation techniques (eg Ficoll gradients).

Particular description regarding the isolation and regulation of endothelial cells (ECs) and in particular endothelial precursor cells (EPCs) can be found, for example, in US Pat. No. 5,980,887 and PCT / US99 / 05130 (WO99 / 45775).

The term “mammal” means primates, breeding or other mammals, such as rodents or rabbits. Preferred primates are chimpanzees, monkeys or human patients in need of treatment. Suitable breeding animals are sand rats, horses, dogs, cats, goats, sheep, pigs, and chickens. Preferred rodents are rats or mice. Preferred BMSCs are isolated from human subjects, such as primates and in particular those in need of cardiovascular therapy.

More particular BMSCs according to the invention are almost spherical when measured by inspection (typically microscope) and also have a diameter of less than about 25 to 35 micrometers, preferably less than about 15 micrometers. Other particular cells of the present invention have an average telomere restriction enzyme fragment length (TRF) of less than about 30-40 kilobases, preferably less than about 20 kilobases, for example about 17 kilobases. Preferred methods of measuring the diameter of the BMSC and determining the TRF are described below.

Another particular BMSC of the invention is essentially euploid, which essentially maintains at least about 10 generations, preferably about 20 to about 200 generations, in the cell medium. Methods of determining cell ploidy are known and are also described below.

More specific BMSCs according to the invention can form endothelial cells (ECs) after contact with EC-promoting conditions as measured, for example, by standard EC differentiation assays. Examples of such cell promoting conditions are known in the art and also include specific angiogen factors and cells as described in US Pat. No. 5,980,887, PCT / US99 / 05130 (WO 99/45775), and references cited therein. Contact with mitogen. These described factors and mitogens include acidic and basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF-1), VEGF165, endothelial growth factor (EGF), transforming growth factors α and β (TGF-α). And TFG-β), platelet-induced growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), erythropoietin, colony stimulating factor (CSF) , Macrophage-CSF (M-CSF), granular leukocyte / macrophage CSF (GM-CSF), angiopoietin-1 (Ang 1) and nitric oxide synthase (NOS); And functional fractions thereof. See Klagsbrun, et al., Annu . Rev. Physiol. 53: 217-239 (1991); Folkman, et al., J. Biol . Chem ., 267: 10931-10934 (1992) and Symes, et al., Current Opinion in Lipidology , 5: 305-312 (1994). Fractions of muteins or mitogens can be used as long as they cause or promote the formation of ECs.

Preferred EC promotion conditions include contact with VEGF, in particular VEGF-1, VEGF165 or both. Further preferred EC facilitation conditions include contact with certain cell matrix proteins such as fibronectin. See Example 2 below.

The expression “standard EC differentiation assay” refers to the assay used to detect and track the action of ECs as described in US Pat. No. 5,980,887 and WO 99/45775. Preferred assays include the search for EC specificity markers as reported in Example 2.

It is also BMSCs capable of forming flexible muscle cells (SMCs) after contact with SMC promoting conditions, particularly as measured by standard SMC differentiation assays. Representative SMC promoting conditions are known and also include contact with at least one angiogen factor or cellular mitogen as described above. Preferred SMC promoting conditions include contact with platelet growth factor (PDGF) comprising a mutein or an active fraction thereof.

The expression “standard SMC differentiation assay” refers to an immunological or molecular test (eg, ELISA, Western blot or the like) that can detect and optionally quantify at least one and preferably all of the following SMC specific markers: αSMA, PDGFβ receptor, SM22α and SMI. PCR). Examples of acceptable assays are described in Example 2.

Another BMSCs according to the invention can form neurons after contact with neuronal cell facilitating conditions, particularly as measured by standard neuronal cell differentiation assays. A variety of neuronal cell facilitation conditions are known and also include the aforementioned factors and mitogens. Contact with one or more. Preference is given to conditions involving the contact of hepatocyte growth factor (HGF) alone or preferably in combination with fibroblast growth factor 4 (FGF-4). Further preferred neuronal cell facilitation conditions include further contact with DMSO or a pharmaceutically acceptable butyrate salt such as sodium or potassium salts. More preferred neuronal cell promoting conditions include contact with appropriate cell matrix proteins such as poly-L-ornithine-laminin.

The term “standard neuronal differentiation” assay refers to the following neurospecific markers: immunization that can detect and optionally quantify at least one and preferably all of GFAP, GalC, NF200, tubulin, myelin basic protein, MAP2, GAD and Tau By chemical or molecular test (e.g., ELISA, Western blot or PCR). See Example 2 (which describes particularly preferred neuronal cell facilitation conditions).

Further suitable BMSCs of the present invention include cells capable of forming cardiomyocytes after contact with accessory cardiomyocytes. The term "adjunct cardiomyocytes" refers to cells that were cardiomyocytes prior to contact with BMSCs. Examples of such ancillary cardiomyocytes include cardiomyocytes that inhabit cardiac tissue as well as fixed cultures of these cells. In some embodiments, the formation of cardiomyocytes by BMSCs is promoted by cell fusion between stem cells and ancillary cardiomyocytes. Such cardiomyocytes (and especially ancillary cells) can be maintained by one or a combination of strategies including strategies including in vitro, ex vivo or in vivo maintenance.

As described above, it is an object of the present invention to provide an implant comprising (or in some cases) isolated bone marrow stem cells described herein. "Graft" means a cell or tissue preparation comprising BMSCs and optionally other cells, such as ECs, SMCs and CMCs, from a mammal. The donor is defined as the source of BMSCs, while the recipient is the recipient of the implant. The immunological relationship between the donor and the recipient can be homogeneous, autologous or heterologous as desired. In a preferred embodiment, the donor and the recipient will be genetically identical and usually the same individual (syngenetic). In this case the implant will be homogeneous to the donor and the recipient.

“Implant” also means BMSCs of the invention administered to a recipient and is also part of one or more tissues or organs of the recipient. Sometimes the word “engraftment” is used to indicate the intended assimilation of BMSCs to target tissues or organs (as BMSCs or differentiated cells). Preferred grafting includes cardiovascular tissues such as arteries, veins and especially cardiac tissue. Implants of the invention may also take the form of tissue cultures that promote differentiation and / or cell replication in which the BMSCs of the invention are combined with other cells and / or mitogen to produce the desired implant. If desired, the formulation can be combined with synthetic or semisynthetic fibers to provide an implant structure. Fibers such as Darkon, Teflon or Gore-Tex are preferred for certain applications.

A particular example of an implant of the invention is the formulation of hBMSCs isolated from a donor to prevent, treat or alleviate the severity of cardiovascular diseases such as myocardial ischemia or infarction. The formulation may comprise a pharmaceutically acceptable carrier such as saline and may optionally also include at least one of mitogen, angiogen factor, CMCs, ECs, EPCs and SMCs to aid in the desired grafting results.

Particular CMCs of the invention express CMC specific protein (cTnl). ECs which are ILB-4 are preferred. Of particular interest are SMCs expressing α-SMA and related markers.

As mentioned above, the present invention also provides a method of making the BMSCs described herein comprising cells (hBMSCs) obtained from a human patient. In one embodiment the method comprises at least one and preferably all of the following process steps.

a) collecting bone marrow cells from a mammal wherein the cells have a size of less than about 100 microns, preferably less than about 50 microns and more preferably about 40 microns or less,

b) culturing (expanding) the collecting cells in medium under conditions of selecting adherent cells,

c) selecting adherent cells and developing these cells in semi-confluency in the medium.

d) diluting the cultured cells sequentially in a chamber having a controlled medium, wherein the dilution is dilution sufficient to form a density of less than about 1 cell per chamber that separates the expanded cells, and

e) incubating (expansion) each of the clonal isolates and then selecting a chamber with expanded cells to create an isolated bone marrow cell population.

More specifically, and in embodiments intended by human cells, hBMSCs can be obtained by taking fresh raw bone marrow (BM) cells from young male donors. Alternatively, such cells can be purchased. Cells are typically isolated from blood cells by centrifugation, hemolysis, and related standard procedures. BMs are washed with an acceptable buffer such as DPBS and filtered to collect cells having a size of less than about 100 microns, preferably less than about 50 microns, more preferably about 40 microns. For example, standard nylon filters can be used. Once isolated, BMs grow on complete culture medium with low glucose (eg DMEM) containing growth factors and abundant sources of cytokines. Fetal bovine serum (FBS) is preferred. Cells are cultured (ie expanded) for less than about 2 weeks, preferably about 1 week or 4 to 6 days. The conditioned medium is then replaced with fresh medium, and adjacent cells are removed from the culture dish and resuspended in fresh medium to select cells that can expand. Selected cells grow semi-confluent (50-90% confluent) and again select adjacent cells. These cells grow in complete medium in tissue culture flasks at a density of about 10 ⁴ cells per centimeter. After the cells reach semi-confluency, they grow (sequentially) in the flask at the same or similar density. The culture preferably continues the selection to expand the cells by passing at least once, typically less than 5 times and preferably about 2 times. Selected cells are continuously diluted into a single well chamber (eg, a standard 96 well plate) at a density of less than about 1 cell per chamber, preferably one half of one cell per chamber. Preferably, the cells are cultured in a controlled medium to promote growth to sub confluence (ie less than 50% confluence). Wells with expanded cell clones are expanded if necessary and plated again.

In particular embodiments the method further explores the following cellular markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117 , Selected cell clones expressing at least one of a CD133, an MHC class I receptor, an MHC class II receptor, or other cellular markers described herein. Methods of making this selection include any of the appropriate assays described herein. In embodiments requiring higher amounts of stem cells, more automated or semi-automated methods are often preferred for luminescent activated cell sorting (FACS) and the like. See the examples described below.

Preferred examples of producing BMSCs are described by Example 1 below.

The invention also provides in one embodiment a method for preventing, treating or alleviating the severity of a heart disease comprising administering to a mammal in need thereof a separate BMSCs, an implant or at least one of the two described herein. To provide. Preferably, administration is sufficient to prevent, treat or alleviate the severity of the disease in a mammal.

In one embodiment, the method further comprises culturing the mammalian cell or implant for at least about 1 week, preferably about 2 to 8 weeks. The incubation period is variable and can be extended or shortened for the health or age of the individual in need of treatment or to treat a particular condition. Representative dosages of BMSCs will depend on these and other recognition parameters, including the disease to be treated and the rate of recovery required. However, for most applications in the range of about 10 ³ to about 10 ⁷ BMSCs, typically about 10 ⁵ of these cells are met. The cells may be administered by certain acceptable routes, including suspending the cells in saline and then administering them with a needle, stent, tether or similar device. In embodiments intended to treat myocardial ischemia or infarction, the administration will be a large pill injection near or directly to the wound site.

Specific methods of regenerating heart tissue following myocardial ischemia are described by Examples 3 and 4. See also Example 5-6 (incorporation of hBMSC in the heart tissue). As mentioned above, the implantation of BMSCs provides a variety of therapeutic effects including raising the level of angiogen factor and increasing capillary and CMS density.

In another embodiment, the method comprises administering at least one angiogen factor or mitogen (or functional fraction of the factor or mitogen) to a mammal in need of treatment. Preferred angiogen factors and mitogens (and methods of use) are described herein as well as in US Pat. Nos. 5,980,887 and WO 99/45775. Alternatively or additionally, the method may comprise administering to the mammal at least one nucleic acid encoding at least one angiogen factor or functional fragment thereof. Methods of administering such nucleic acids to mammals are described, for example, in US Pat. Nos. 5,980,887 and WO 99/45775. Treatment with angiogen factor / mitogen proteins or nucleic acids encoding them prioritizes the use of BMSCs or can be used during or after such treatment if necessary.

In another embodiment, the method further comprises administering to mammalian endothelial progenitor cells (EPCs). Embodiments of the invention find use in need of good blood vessel growth, particularly for treating cardiovascular disease. A method of making and using EPCs is described. See, eg, US Pat. No. 5,980,887. Exemplary methods may include separating EPCs from mammals and contacting the EPCs with at least one angiogen factor and / or mitogen degenerate.

As described above, the present invention is applicable to the prevention and treatment of a wide range of cardiovascular diseases including congestive heart disease (CHF), ischemic cardiomyopathy, myocardial ischemia, and infarction. If desired, these methods include, for example, ultrasound cardiac examination, ventricular end cardiac dilation dimensions (LVEDD), end-heart contraction dimensions (LVESD), fractional shortening (FS), wall motion score index (WMSI) and LV cardiac contraction pressure. Examining cardiac function in a mammal in need of treatment by examining at least one of the (LVSP). Preferred inventive methods, including the prevention or treatment of particular cardiovascular diseases, will exhibit good cardiac function as exemplified by one or more of these tests. The term "good" means at least 10% improvement, preferably at least 20% or 30% over a control that has not received the invention composition (or has received a placebo). Specific methods of carrying out these tests are known and described in the Examples section.

The present invention provides for example a method for separating cells from the following components: BMSCs, preferably hBMSCs and optionally mammals; Grafting with the implant described herein, preferably hBMSCs or cells or tissues derived therefrom and optionally the direction of making, maintaining and / or using the implant; Further provided are pharmaceutical products that prevent, treat or alleviate the severity of heart disease, including at least one of cell culture, tissue or organ, and optionally the direction of making the same. In one embodiment the product comprises at least one angiogen factor, mitogen; Or functional fractions thereof. In another embodiment, the product further comprises at least one nucleic acid encoding angiogen factor, mitogen or a working fraction thereof.

As also described above, the present invention provides isolated BMSCs such as hBMSCs prepared by the methods described herein. Such cells have advantages such as the ability to proliferate long term without desirable plasticity and significant culture problems (eg, unwanted drainage and pluripotency loss). In one embodiment, such cells comprise the following cell markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, Cells are prepared by collecting cells that do not express navigable levels of at least one of an MHC class I receptor, an MHC class II receptor, or other markers described herein.

The present description particularly shows that the invention can be used to isolate BMSCs and in particular hBMSCs with potentially unlimited proliferative capacity from single cells and also allows for triple pass differentiation including ECs, SMCs and CMCs. Implantation of hBMSCs in acute infarcted myocardium attenuates heart disorder by new differentiation into myocardial tissue and favorable paracrine effects on adjacent cells.

More specifically, the present invention, including the examples, shows that novel stem cell (SC) populations have been identified in adult human bone marrow (BM). Further shows the isolation and proliferation of these cells at the single cell level without apparent senescence or loss of pluripotency for> 140 population doubling (PDs). These colony expanded human BM-derived pluripotent SCs (hBMSCs) have minimal to unsearchable expression of CD90 and CD117. Thus, the hBMSCs isolated by the inventors do not belong to known BM-derived SC pops such as hematopoietic SCs, mesenchymal SCs or pluripotent adult progenitor cells. hBMSCs show the potential for differentiation (Diff) into three layers in vitro. Coculture of neonatal rat cardiomyocytes (CMC), rat aortic endothelial cells (EC), or rat flexible muscle cells (SMC) with hBMSCs showed a phenotypic change consisting of calm differentiation and cell-to-cell fusion. We tested whether acute infarcted myocardium (Myoc) can be recovered by transplantation of hBMSC (Xplnt). Immediately after inducing MI in nude rats, hBMSCs are xplnt'd into the wall of the surrounding infarct area. Muscle function measured by signal and pressure transducers is significantly better in hBMSC xplnt rats after 28 days than in total BM cell xplnt or saline injected control rats. Healthy grafting of xplnt'd cells was observed, indicating Diff in CMCs, ECs and SMCs. In hBMSC transplantation Myoc, multiple angiogen cytokines and essential cardiac transcription factors were significantly overregulated and also higher proportions of EC and CMC were observed. CONCLUSIONS: The new pluripotent SC population is present in human BM and locally xplnt'd hBMSCs increase the proliferation and preservation of host cardiomyocytes as well as improve the outcome of acute MI by new angiogenesis and muscle formation. This allows human BM-derived SCs to differentiate into all the elements required for regeneration of ischemic myocardium in vitro and in vivo.

As described above, the present description shows that a novel adult human stem cell population, i.e., hBMSC, which does not belong to a known population of adult stem cells, has been isolated starting from a single cell level from mixed total BM cell cultures and also has a loss of plasticity or proliferation of senescence. Expansion by clonal in culture of> 140 PDs without initiation. Clonal induced hBMSCs differentiated into cells of three fungal layers in vivo (endoderm, mesoderm, neuroectoderm). Transplantation of hBMSCs into an animal model of MI improves functional and pathological changes following MI. In addition, the mechanism of improved cardiac action not only consists of differentiation into essential cardiomyocytes such as CMCs, ECs and SMCs, but also involves the apparent paracrine effect of transplanted stem cells, which stimulates host cardiomyocytes and ischemic wounds. To prevent apoptosis of dangerous cells.

The hBMSCs we have identified are a unique population of adult BM derived pluripotent SCs in that <1% of hBMSCs express CD90 and CD117. All common marker panels that define HSC, MSC and MAPC do not conform to the profile of hBMSC. HBMSC does not express the well-known MSC marker proteins, CD105 (SH2) and CD73 (SH3 and SH4), although the medium used is similar to that of MSC culture {Barry, 1999; Barry, 2001}. The absence or peak expression of surface molecules is a prerequisite for the plasticity of hBMSCs as shown in other adult pluripotent stem cells [Jiang, 2002] Colter, 2001]. The unique surface phenotype of hBMSCs arises from the use of total BM cell populations for initial culture and cronal separation / selection procedures. The present invention addresses one important problem in stem cells, namely whether pluripotent stem cells can be culture-expanded from a single cell. Here we found for the first time that ex vivo and in vivo differentiation of hBMSCs from mesoderm, ectoderm and neuroectoderm occurs from clones developed from single cells. In contrast to MAPCs, hBMSCs do not express genetic markers of ESCs, such as Oct-4 and Rex-1, which are believed to be essential elements for MAPCS. The study of the present invention suggests that factors including maintenance of undifferentiated state and unrestricted proliferative potential of adult SCs are different from factors of ESCs. Another difference is the fact that BMSCs did not require leuchemia inhibitory peptides of culture expansion [Yoon YS, 2002], which are essential for MAPC culture. Rather, maintenance of low cell density plays an important role in maintaining the pluripotency and expandability of hBMSCs.

Recent studies have demonstrated an important role for cell fusion of stem cell plasticity. This in vitro data indicates that fusion and trans-differentiation are involved in phenotypic change of hBMSCs into ECs, SMCs and CMCs, and its dissemination depends on cell type. Although no exact studies have been performed in vitro to quantify the contribution of cell fusion to the observed phenotypic changes, the results of in vitro studies suggest that fusion and trans differentiation may play a role. Whatever the contribution from each component, the transplanted hBMSCs assisted the fused hepatocytes to reconstruct myocardial integrity after ischemic wounds as in metabolic liver disease. From this study it is speculated that cell fusion is not necessarily an important barrier to therapeutic applications, especially for adult SCs.

Compared to other SCs, the hBMSCs of the present invention provide an advantage for regenerative therapies of heart disease. Stem cells shown to improve heart function include EPCs, CD34 (+) cells, and hemangioblasts, which have been shown to promote neovascularization in vivo. Pluripotent stem cells such as MSCs, c-kit positive passage negative cells, SP cells, and ESCs were tested in an ischemic model. ESCs did not demonstrate the potential to differentiate into CMCs, but did not appear to differentiate into ECs or SMCs in animal models of IHD. Moreover, the use of ESCs is hampered by certain ethical issues that must be addressed before clinical application. Trans-differentiation of SP cells in multiple passages in IHD has been demonstrated in human cells as well as mouse cells, and the therapeutic effect is unknown. Mouse but human Lin (-) c-kit (+) cells also show pluripotency and therapeutic effects, but lack of these SC or BM in circulation and inability of culture-expansion limit their therapeutic use. Human MSCs have been shown to trans-differentiate into CMCs but their morphological appearance is different from that of organized CMCs, and EC and SMC differentiation have not been demonstrated. In contrast, the hBMSCs described herein have the versatility necessary in vitro and in vivo to regenerate damaged myocardium, are culture-expandable and also describe the functional capacity of therapeutic applications.

Coronary heart disease represents 50% of all cardiovascular deaths and almost 40% of heart disease frequency. The present findings provide compelling evidence that the invention can be used to prevent, treat or alleviate the severity of various heart diseases including diseases associated with infarction and / or ischemia. More specifically, the present invention promotes new angiogenesis and also enhances angiogenesis and proliferation of current CMCs, reduces progressive fibrosis and also increases ventricular function in rodent models of MI. We believe that this may promote successful treatment of acute MIs during the course of ischemic tissue damage by adult human stem cells. The present invention is also applicable to the prophylaxis or treatment of IHD, in particular to increasing the mid to long term and long term results of IHD.

      The following examples are illustrative and do not limit the invention. All documents described herein are incorporated by reference.

Example  One

Clonal, surface epitopes, euploidy and proliferation of hBMSCs were evaluated as follows. New raw human BMs were purchased from young male donors. Three different bone marrow specimens from three different donors for SC culture were used. After a series of cultures of total bone marrow cells in plastic dishes in Dulbecco Modified Eagle Medium (DMEM) with low (1 g) glucose containing 17% of fetal bovine serum (FBS), cells were labeled with a red fluorescent dye, DiI. . After limiting dilution (1-2 cells per well in a 96 well plate) single cells visualized by luminescence microscopy were selected. In wells containing single cells (FIG. 1A), 6 ± 4% (range 2-13%) demonstrated cell survival and proliferation. When cells grow 4--50% confluent, cells from each well (one clone) grow native to one well of a 6-well plate and thereafter 4-8 × 10 ³ cells / cm ² . At the density were grown in line in 25 cm ² tissue culture flasks (T25), T75 and T175, respectively. Thereafter, cells were incubated at a density of 4-8 × 10 ³ cells / cm ² in T175 and repeated 1: 20-40 dilutions. Reseeding was performed three times and the fastest growing clones were selected in every culture and expanded in a series of cultures. After at least 10 clones were obtained from each bone marrow in Generation 6, two clones were selected for continuous culture. Morphologically, compared to MSCs, hBMSCs are more spherical cells, smaller in size (diameter <15 μm) and also exhibit a high nuclear-to-cytoprism ratio (FIG. 1). Clonal cell lines derived from this method performed over 140 population doublings (PDs). The doubling time is 38 ± 9 hours at 20-80PDs. FACS analysis with multiple epitopes demonstrated minimal to zero expression (0-1%) of CD90 (FIG. 1C) and CD117 (FIG. 1S). hBMSCs did not spontaneously differentiate and also remained phenotype during culture expansion. In contrast, hBMSCs cultured prior to clonal isolation expressed low levels of CD105, CD90 and CD117, while mesenchymal stem cells expressed high levels of CD29, CD44, CD73, CD105 and CD90 (FIG. 1B). Major histocompatibility complex (MHC) class I (ABC) and II (DR) molecules and known hematopoietic stem markers (CD34, CD133, FLK-1, Tie2) were not expressed (FIG. 1S). RT-PCR analysis is negative for Oct4 and Rex-1, which are known markers of fetal stem cells and MAPCs. These results indicate that hBHSC does not belong to known HSCs, MSCs or MAPSs and is also immunologically inactive. The average telomere restriction enzyme fragment (TRF) length cultured for 5PDs is about 17 kilovas (kb); When retested after 120 PDs, the mean TRF remained unchanged (FIG. 1D). DNA ploidy (number of DNA copies) was examined by FACS analysis after contaminating DNA with iodide propidium. HBMSCs cultured for 20 and 140 PDs from three different clones demonstrated no evidence for increased ploidy, suggesting that euploidy is maintained during culture-expansion.

1A-E are discussed in more detail as follows. a . Phase contrast and luminescent images show single cells per well. b . Morphologically, many hBMSCs exhibit rounded shapes with cell sizes of <15 μm in diameter. Scale bar = 50 μm c . Clonal isolated hBMSCs cultured for 140PDs were labeled with PE or FITC-binding antibodies against human CD29, CD44, CD73, CD105, CD90 or immunoglobulin isotype controls. Cells were analyzed by FACStar flow cytometer (BD). Indigo ray, control immunoglobulin; Red line, specificity Ab. HBMSCs cultured prior to clonal isolation expressed low levels of CD105, CD90.

Clonal isolated hBMSCs showed only CD90 with zero to minimal expression (0-1%) (FIG. 1C). In contrast, purchased mesenchymal stem cells expressed high levels of CD29, CD44, CD73, CD105 and CD90. d . Average telomere restriction enzyme fragment (TRF) length of hBMSCs cultured for 10PDs (lane 1, kb) and 120PDs (lane 2, kb) indicating no difference in mean TRF. e . DNA ploidy analysis. HBMSCs were contaminated with propidium iodide before and after clonal isolation and also subjected to FACS analysis. HBMSCs cultured for 20 (left panel) and 140 (right panel) PDs demonstrated <1% of diploid abnormality DNA content. Representative Example of> 3 experiments.

Example  2: hBMSC Plasticity of: In vitro  differentiation

The in vitro differentiation potential of hBMSCs was tested by employing or modifying previously published culture conditions of adult and fetal stem cells that primarily require passage-specific cytokines. To induce differentiation into ECs, hBMSCs were treated with 2% FBS, 10 ^-8 dexamethasone and 10 ng / ml VEGF and 5 x 10 ⁴ cells / in 0.1% gelatin or fibronectin coated glass chamber slides in DMEM or EBM-2 (clonetic). Replanted in cm ² . After 5 days of culture, hBMSCs formed a vascular-like structure (FIG. 2A, left top panel). After 14 days of culture, hBMSCs expressed an EC specific phenotype such as the new Willebrand factor (vWF), Flk1, VE-cadhedrin, CD31, human-specific EC marker Ulex Europaeus type 1 (UAE-1) (FIG. 2a). On day 14, 63 ± 8% (VE-cadherin) to 85 ± 12% (UAE-1) demonstrated the EC phenotype by immunocytochemistry. RT-PCR, VE-cadherin, CD34, Flk-1, Tie and CD31 of EC specific genes also confirmed the differentiation of hBMSC (pre-differentiation) into EC phenotype (post-differentiation).

To induce differentiation in SMC passages, hBMSCs were administered in 1 × 10 cells / in uncoated or fibronectin coated plastic dishes in 1-2% DMEM or EBM-2 supplemented with PDGF-BB (50 ng / ml, R & D). implanted in cm ² [Hellstrom, 1999; Yamashita 2000]. After 14 days of culture, 89 ± 6% and 67 ± 12% of hBMSCs were positively contaminated with α-flexible muscle actin (α-SMA) and calponin, respectively, with differentiation into the SMC phenotype (FIG. 2C). RT-PCR identified new expression of SMC specific genes, αSMA, PDGFβ receptor, SM22α, and SM1 (FIG. 2D).

2A-D are described in detail as follows. a . Huffman images (left upper) 5 days after incubation with DMEM in a gelatin-coated glass chamber in which hBMSCs formed a representative vascular-like structure. Immunofluorescence images demonstrate that EC-specific proteins such as vWF, Flk1, VE-Cadherin, CD31 and UEA-1 after 14 days of culturing hBMSC in EC differentiation medium. b . RT-PCT analysis using EC specific gene primers, VE-cadhedrin, CD34, Flk-1, Tie2 and CD31 confirmed the differentiation of hBMSC (pre-differentiation) into EC phenotype (post-differentiation). c . HBMSCs cultured in 2% DMEM containing PDGF-BB for 14 days demonstrated the expression of SMC specific protein, α-SMA, and calponin by IF contamination. d. RT-PCR analysis shows that the SMC specific genes, PDGFR-β, α-SMA, SM22α and SM1, are only expressed after induction of differentiation. Thick ladder of size marker at RT = PCR (b and d) = 600 bp.

To induce neuronal differentiation, hBMSCs were combined with 100 ng / ml bFGF, 20 ng / ml EGF, and B27 supplements in DMEM / F12 on 4 x 10 ⁴ / cm on plastic dishes or poly-L-ornithine-laminin coated dishes. Plates were cultured at ² [Palmer, 1999; Mezey, 2000; Brazelton, 2000]. After 10-14 days, hBMSCs showed the morphological and phenotypic characteristics of various neural passage cells (FIG. 3A). Immunofluorescence cytochemistry revealed astrocytic cells (glyal acid fibril protein, GFAP), proliferative glial cells (galactoserebroside, GalC) in hBMSCs of 22 ± 7%, 15 ± 6%, 57 ± 9% (NF200). ), And the expression of phenotypic markers of neurons (neufilament 200, NF200; β-tubulin III) isoform, β-tubulin III). RT-PCR has a variety of neural pathway specific genes such as GFAP, myelin basic protein (MBP, prostatic glial cells), MAP2 (microtube-binding protein 2, neurons), GAD (glutamic acid decarboxylase, neurons), and Tau New expression of (neurons) was confirmed [Sanchex-Ramos, 2000].

to see if hBMSC could differentiate into endodermal passage, hBMSC 10 ^{- 8} a 2% FBS 1% matrigel, 25ng / ml, 10ng / ml FGF-4 and 10mg / ml DMSO or 0.5mM supplemented with dexamethasone acid Plated at 3-4 x 10 ⁴ cells / cm ² for sodium [Shen, 2000; Hamazaki, 2001; Oh, 2000; Schwartz, 2002]. After 10-14 days of culture, about 60% of hBMSCs obtained endothelial form. IF histochemistry showed expression of endoderm / hepatocyte genes, HNF 3β and αFP at 10 days in cultured hBSCs, and HNF 1α and CK18 at 14 days (FIG. 3G-1). RT-PCR analysis revealed new expression of endoderm passage specificity genes, CK18, CK19, αFP and albumin (FIG. 3M).

3A-M are described in more detail as follows. a . Morphological characteristics of hBSCs in neuronal differentiation cultures showing representative appearance of neurons. Scale bar = 50 μm. be . IF contamination of hBMSCs after neuronal differentiation was characterized by multiple specific proteins: GFAP (astrocytic cells), GalC (protuberant glial cells), NF200 (neurons), and β-tubulin III. f. RT-PCR confirms new expression of neural subband-specific genes, GFAP, MBP, progenitor glial cells, MAP2 (neurons), GAD (neurons), and Tau (neurons) after induced neural differentiation. Phenotypic characterization of hBMSCs into endoderm (parietal epithelial) cells. IF localization of HNF 3β (FITC) and αFP (Cy3) and CK18 (FITC) and HNF 1α (Cy3) on day 10 (gi) in hBSCs cultured on Matrigel with medium containing HGF on day 10 (gi) anger. m. RT-PCR confirms new expression of endoderm passage-specific genes, CK18, CK19, αFP and albumin after induced neuronal differentiation.

Example  3: newborn Rat Into cardiomyocytes (NRCM) Coculture  after BNSC Myocardial differentiation

To induce myocardial differentiation, hMSCs were cocultured with neonatal rat cardiomyocytes (NRCM). NRCMs were plated at 1 × 10 ⁵ cells / cm ² and incubated in DMEM (low glucose) containing 10% fetal calf serum (FCS). On day 3 hBMSCs labeled with DiI were added to the culture NRCM in a ratio of 1: 4 and incubated for up to two weeks [Condorelli, 2001]. After fixing and contaminating with antibodies to cardiac specific markers, immunoluminescence images were obtained. DiI-labeled hBMSCs (FIG. 4B, f, j) show red luminescence from DiI labels and also CMC specific proteins such as cardiac troponin I (cTnl), atria natriuretic peptide (ANP), α-myosin heavy chain Cells positive for (α-MHC) show green (Figure 4C, g, k). Binding images illustrate the fraction of DiI-labeled hBMSCs, which were also positively contaminated with CMC specific proteins showing that a subpopulation of hBMSCs characterizes the CMC phenotype in coculture (FIG. 4). MRNA expression of cardiac graft factor was assessed by RT-PCR (FIG. 4M). Prior to coculture, GATA-4 and Nkx2.5 were not expressed in nBMSC (left lane) or NRCM (center lane) cultures. Co-culture of hBMSC and NRCM (right lane) induced new expression of GATA4 and Nkx2.5. GATA-4 is a cardiac specific electron factor known to activate phenotypes of various cardiac genes, such as myosin sun chain, TnT, TnI and α-MHC and ANP. Nkx2.5 is an early phase of CMC differentiation and is a limited transcription factor [Srivastava, 2000; Bruneau, 2002]. These findings therefore suggest that hBMSC has entered a differentiation generation towards the CMC phenotype. Caution, CMC differentiation of hBMSCs was observed when they were cocultured with conditioned media from NRCM.

As shown in Figure 4i, NRCM and hBMSC are tightly coupled to each other. Therefore, the possibility of fusion cannot be determined by the above experiment. Moreover, recent studies have reported that phenotypic changes in ESCs and bone marrow induced stem cells may occur as a result of cell fusion [Ying, 2002 Terada, 2002]. In order to determine whether this mechanism is the basis for hBMSC differentiation, CFDA-SE (carboxyfluorescein diacetate succinimidyl ester) -labeled NRCM and DiI-labeled hBMSCs are used as adjacent cells. It was used for coculture because it was not moved. After 7 days of coculture, cultured cells were fixed and contaminated with cTnI followed by AMCA-binding secondary antibody (blue luminescence) (FIG. 4P). In order to avoid cell overlap, we selected a region of a single cell layer to image the assay. Specific DiI-labeled hBMSCs, indicated by arrows, as shown in FIG. 4N-Q, were also positive for green (NRCM) and indigo blue (cTnI), indicating that cell fusion occurred. Truly differentiated cells were exemplified by red and indigo fluorescence positive and green luminescence negative (FIG. 4 o-q, arrow head). These results indicate that both differentiation and fusion occur under the same culture conditions. After counting 5000 cells per coculture from three different experiments, the dominance of differentiation and fusion was 2.9 ± 1.1% and 3.2 ± 0.9%, respectively. To determine the occurrence of fusion between hBMSCs and ECs / SMSs, rat aortic endothelial cells (RAECs) or rat vascular flexible muscle cells (RVSMCs) were each co-cultured with hBMSCs. When RAECs and RVSMSs reached 50-60% confluence, cells were labeled with CFDA-SE (green fluorescence). After 2 days, DiI-labeled hBMSCs were added to the culture plates of RAECs and RVSMCs in a ratio of 1: 4 and incubated for 7 days. The cultured cells were then fixed and contaminated with VE-tardherin or α-SMA followed by AMCA-binding secondary antibody. Using the above criteria of fusion and sedation differentiation under luminescence microscopy, the predominance of fusion was 5.3 ± 1.1% for ECs and 7.4 ± 0.9% for SMCs, while the predominance of differentiation was 3.4 ± 0.7% and 3.6 ± 0.9% . These results indicate that the phenotypic change in hBMSC consists of fusion and differentiation, and therefore immunohistochemistry using passage specificity marker proteins that define differentiation may overestimate the true prevalence of differentiation in vivo. In Vivo Fusion and Differentiation This ratio may differ from those recorded in detail in vitro.

4A-M are further described as follows. am . Co-culture of hBMSC with NRCM for investigation of cardiac differentiation. Primary NRCMs were isolated from F344 rats and incubated with DMEM. On day 4 DiI-labeled hBMSCs were added to the culture NRCM at a ratio of 1: 4 and incubated for up to two weeks. IF images show cocultured DiI-labeled hBMSC (b, f, j) in red and NRCM contaminated with CMC specific protein, cTnI (c), ANP (g) and α-MHC (k) in green . Dual luminescent positive cells in the binding images (d, h, i) indicate that a subpopulation of hBMSCs displays the CMC phenotype (arrow). Note: some hBMSCs remain untransdifferentiated (arrow ends b, d, j, l). DAPI nuclear counter-contamination (a, e, i) indicates no nucleation overlap, suggesting that this apparent trans-differentiation does not result from a simple overlap of hBMSCs and NRCMs. MRNA expression of cardiac transcription factors was analyzed by RT-PCT (m). Prior to co-culture, cardiac transcription factors GATA4 and Nkx2.5 were not expressed in hBMSC (lane 1) and NRCM (lane 2). Coculture (lane 3) induced new expression of GATA4 and Nkx2.5. nq . Co-culture of pre-labeled NRCMs and hBMSCs for investigation of cell fusion. To determine whether the fusion of two cells is based on a change to the CMC phenotype, we co-cultured dNRCMs labeled with green luminescent dye, DFCA-SE (n) and DiI-labeled hGMSC (o). After 7 days of coculture, cells were contaminated with cTnI followed by AMCA-binding secondary antibody (blue luminescence) (p). The triple color positive cells (arrows) in the IF image (nq) are fusion cells expressing cTnI. HBMSCs trans-differentiated with CMC exemplify red and blue luminescence positive and green negative (end arrow). rx. To determine the contribution of fusion and differentiation to the phenotypic changes of ECs (ru) or SMCs (vy) of hBMSCs, CDFA-SE-labeled rat aortic endothelial cells (RAESc) or rat vascular flexible muscle cells (RVSMCs) were 4: 1. Co-cultured with DiI-labeled hBMSC at a ratio of 7 and cultured for 7 days. After fixation, cells were contaminated with VE-cadherin or α-SMA followed by AMCA-binding secondary antibody (blue luminescence). Arrow ru represents the fusion between RAECs (green) and hBMSCs (red) expressing VE-cadherin. Arrow end r- u illustrates the ECs differentiated from hBMSCs. Arrow vx indicates fusion between RVSMCs (green) and hBMS (red).

Example  4: acute ischemia In the myocardial layer  Regeneration effect

We tested whether the ischemic obstructive myocardial layer can be recovered by transplantation of hBMSC as follows.

Immediately after corona binding in nude rats, 8 × 10 ⁵ hBMSCs were implanted into the wall of the surrounding infarct area. Equal number of human total bone marrow cells (TBMC) and equal volume of PBS were used as controls. Of the 15 rats undergoing surgery, 12 rats survived in the TBMC and PBS groups during the 4 week study, while 14 rats that received hBMSC survived. To evaluate cardiac function, echocardiography was performed before and 4 weeks after surgery. Baseline left ventricular end-heart dilation and end-heart contraction dimensions (LVEDD, LVESD) fraction shortening (FS) and wall motion score index (WMSI) were similar among rats receiving hBMSC, TBMC or PBS (data not shown). Ultrasonography performed 4 weeks after treatment was significantly smaller in rats receiving hBMSC than both LVEDD and LVESD treated with TBMC or PBS (LVEDD, p <0.05 vs TBMC and PBS; LVESD, P <0.01 vs. TBM and PBS) (Figures 5a, b), thus the FS was significantly larger (p <0.01 vs TBMC and PBS) (Figure 5c). The wall motion score index (WMSI), which indicates the degree of regional wall motion abnormality, was significantly better (P <0.01 vs TBMC and PBS) (FIG. 5D). LVEDD, LVESD, FS and WMSI did not differ in TBM and PBS treated rats. Attention, the frequency of dyskinesia was 14% vs 58% and 67% in hBMSC vs. TBMC and PBS treated rats (P <0.05). Invasive hemodynamic measurements showed that LV heart contraction pressure (LVSP), + dP / dt (maximum pressure rise rate) and -dP / dt (minimum pressure drop rate) were all significantly greater in hBMSC transplanted rats than in TBMC or PBS injection rats (All p <0.01 each) (FIG. 5E, f). Together, these data show that hBMSC transplantation results in increased functional recovery and more favorable remodeling following myocardial infarction.

5A-D are described in more detail below. ad . Four weeks after MI and cell transplantation, echocardiography showed smaller LVEDD, LVESD and better FS and WMSI in hBMSC transplanted rats compared to TBMC and PBS treated rats, indicating improved cardiac function. WMSI: Wall Movement Score. ef . Invasive hemodynamic measurement using mila catheter 4 weeks after hBMSC implantation. LV heart contraction pressure (e) and + dP / dt and -dP / dt were significantly increased in hBMSC transplanted rats compared to the control groups. ^* P <0.05, ^** P <0.01,

Example  5: In vivo  transplantation hBMSC Grafting and multi-passage trans-differentiation

To determine the extent and size of the implanted hBMSC graft, the 4-week-myocardial site was examined from frozen samples. Under luminescence microscopy, the incorporation of a number of DiI-labeled BMSCs in peripheral infarcts and infarct sites was observed (Figure 6A, b). In contrast, the TBMC transplanted heart shows a smaller number of dispersed DiI-labeled cells located mostly in the infarct site. Immune phenotypic characterization showed that the grafted DiI-labeled hBMSCs (red) were positively contaminated with CMC specific proteins, cTnI and ANP (green) (α-MHC data not shown) and also distinguished from host CMCs at peripheral infarcts and infarct sites Formed clumps of morphologically mature CMCs that could not be formed (FIG. 6, e-1). This represents CMC differentiation. Differentiation of hBMSCs into ECs and SMCs was confirmed by co-localization of transplanted hBMSCs with ILB4 positive vascular ECs and α-SMA positive SMCs, suggesting differentiation into EC and SMC phenotypes (FIG. 6, m-t). CMC differentiation only in hBMSC transplanted rats. Differentiation into CMC, defined by morphological similarity and CMC specific protein expression (cTnI), was observed to be detectable by 12 of 14 surviving hBMSC transplanted rats (85%). Binding masses regenerating CMCs similar to normal host myocardium were found in 9 of 14 rats (64%) and only dispersed cell differentiation was observed in 3 other rats, as shown in FIGS. 6H and 8C. (21%). However, CMC specific protein expression was found in all hBMSC treated rats. EC and SMC differentiation, defined positive by morphology and marker, were found in all rats examined. Together, these findings suggest that transplanted hBMSCs are strongly grafted to infarct / ischemic myocardium, survive for a delayed period of time, and also produce new muscle and angiogenesis. Caution, pathological examination indicated no teratoma, angiomatosis or bone formation in the hBMSC transplanted heart.

6A-D are described in more detail as follows. Incorporation of DiI-labeled hBMSCs and TBMCs into infarct myocardium. A number of hBMSCs (red fluorescence) (a) are grafted to the infarct and peripheral infarct areas of myocardium 4 weeks after transplantation. On the other hand, significantly less TBMC (red luminescence) is mostly present in the infarct region (a). (b) and (d) are Hoffman images of (a) and (c) showing localization of grafted cells. el . Immune Phenotypic Characterization of Differentiated hBMSCs into CMCs. 4-week myocardial samples were contaminated with cTnI (g) and ANP (k) (searched as FITC-labeled secondary Abs, respectively). Transplanted DiI-hBMSCs are positively contaminated with two markers and are indistinguishable from host CMCs, which also indicates the regeneration of clumps of CMCs. mp . Introduction of hBMSC to Vascular ECsso. Myocardial sites, EC markers contaminated with ILB4 (o), demonstrate that DiI-hBMSC co-localizes with vascular ECs suggesting trans-differentiation into ECs (arrow). qt . Introduction of hBMSCs into Vascular SMCs. Myocardial sites contaminated with α-SMA (s) illustrate the co-topics DiI-hBMSC with vascular SMCs, suggesting differentiation with the SMCs phenotype (arrows s, t).

Example  6: Transplantation Increases Proliferation and Survival of Host Myocardium hBMSC

Since the aforementioned physiological effects of hBMSC transplantation cannot be explained in some of the rats by the degree of multiple passage differentiation therein, the inventors have discovered whether the transplanted hBMSCs affect the proliferation and survival of host cardiomyocytes. I wanted to pay. In order to measure the proliferative fraction of cardiomyocytes, the small-osmotic pump constantly applies 5-bromo-2'-deoxyuridine (BrdU) for 4 weeks to label all cells entering S phase of the cell cycle. Transplanted to feed. At the site from the hBMSC transplanted heart, multiple proliferating cells were observed in both host cardiomyocytes and transplanted cells (FIGS. 7A-D). In contrast, significantly fewer BrdU-positive cells were observed in TBMC or PBS injected hearts (FIG. 7E, f). The BrdU index, ie the percentage of BrdU positive nuclei relative to the total number of nuclei counted at each site, was significantly higher in the hBSMC transplanted heart than the control (hBMSC vs TBMC, PBS; 25.8 ± 5.2% vs 11.2 ± 3.1%, 9.5 ± 2.8% , P <0.001). Dual IF histochemistry using BrdU and mAb for ILB4 or cTnl indicated that CMCs and ECs were positive for BrdU (FIG. 7G-n). The BrdU indices of ECs and CMCs were again higher in bBMSC transplanted hearts (vs TBMC, PBS; ECs, 8.3 ± 2.7 vs 2.9 ± 1.5, 2.4 ± 1.3, P <0.01; CMCs, 2.1 ± 1.2 vs 0.2 ± 0.1, 0.2 ±) 0.1, P <0.01). These results indicate that transplanted hBMSCs increase proliferation of host cardiomyocytes, including ECs and CMCs.

Next, the inventors investigated whether or not it could affect the apoptosis process, which is generally considered one of the major mechanisms involved in ongoing myocardial degeneration after acute MI. TUNEL evaluation was performed using 1 week myocardial sample (n = 4). Apoptosis index, ie the percentage of TUNEL (+) nuclei relative to the total number of nuclei counted at each site, was three times lower in the hBMSC transplanted heart (apoptotic index vs TBMC, PBS, 2.1 ± 0.8 vs 6.6 ± 1.2, 7.3 ± 1.1 , P <0.001) (FIG. 7o-q). These differences are particularly evident in the hyperinfarct region. The apoptosis index of CMCs and ECs obtained after α-sarcomer actinin or incidental contamination of ILB4 and TUNEL evaluations was significantly reduced in hBMSC transplanted heart (vs TBMC, PBS; CMCs, 0.5 ± 0.1 vs 2.1 ± 0.3, 1.93 ± 0.3, P <0.001; ECs, 0.7 ± 0.1 vs. 2.5 ± 0.3, 2.0 ± 0.2, P <0.01) (Figure 7r-u). Thus, while hBMSC transplantation promotes survival of dangerous CMCs and ECs, an alternate mechanism that can preserve myocardial function is revealed by these studies.

7A-U are described in more detail as follows. ah . Identification of proliferative cells by BrdU immunohistochemistry. In the hBMSC transplanted heart (ad) portion, multiple BrdU positive cells (green dots) were observed in both (c) host cardiomyocytes and transplanted cells. In contrast, fewer BrdU positive cells were observed in TBMC (e) or PBS (f) injected rat hearts. gj. Simultaneous contamination with antibodies to BrdU (h) and ILB4 (i) demonstrates the presence of BrdU positive cells (arrows) in capillary ECs of the hBMSC transplanted heart. en . Concurrent contamination with antibodies to BrdU (h) and cTnI (m) demonstrates the presence of BrdU positive cells (arrows) in mature CMCs (n) of the hBMSC transplanted heart. mo . Concurrent contamination of anti-α-sarcomeric actinin mAb and TUNEL (n) or PBS (o) -treated myocardium to identify apoptotic cardiomyocytes (arrows). Less apoptotic cells are evident in the peripheral infarct area of rats receiving hBMSC (o) compared to those receiving TBMC (p) or PBS (q). Magnification x 400. rs . Representative images obtained after contaminating with mAbs for α-sarcomeric actinin (red) and TUNEL (green) demonstrate CMC cell death (arrows) from PBS injected hearts. tu . Representative images obtained after contaminating with ILB4 (red) and TUNEL (green) demonstrate EC apoptosis (arrows) from PBS injected hearts.

Example  7. Implanted hBMSC of Paraclean  effect; Angiogen Cytokines  And excessive regulation of cardiac transcription factors

VEGF-A, by semi-quantitative RT-PCT using cardiac samples harvested 14 and 28 days after hBMSC or PBS treatment, to identify potential paracrine mechanisms responsible for the therapeutic effect of hBMSC after myocardial wounds Angiogen cytokines such as angiopoietin-1 (Ang-1) and 2 (Ang-2), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-B (PDGF MRNA expression of cardiac transcription factors such as -B), transforming growth factor (TGF) -β and Nkx2.5, GATA-4 and MEF2c were evaluated. Expression of all tested angiogen cytokines was significantly regulated at both 2 and 4 weeks in hBMSC transplanted heart compared to the PBS group (P <0.01). Moreover, high expression of all angiogen cytokines was maintained at 4 weeks in the hBMSC transplanted heart. These findings suggest that transplanted hBMSCs can regulate the expression of multiple cytokines with the potential to promote angiogenesis as well as angiogenesis. GATA-4, Nkx2.5 and MEF2c were markedly regulated in the hBMSC transplanted heart compared to the control (FIG. 8B), which would increase muscle formation by transplanted hBMSCs to increase host CMC differentiation and / or directly differentiate into new CMCs. Imply that it can. Overall, the overregulation of multiple factors may partially explain the proliferative effects of transplanted hBMSCs on host ECs and CMCs.

8A-B are described as follows. Tissue samples were harvested 2 and 4 weeks after hBMSC or PBS treatment and mRNA expression was also examined by semiquantitative RT-PCR. Representative gel photographs of RT-PCR products (n = 4), and quantification of mRNA fragments (right panel) of angiogen cytokines (a) and cardiac transcription factors (b) based on GAPDH expression. Caution: Significant transient regulation of all factors in hBMSC transplant samples. The experiment was performed at least three times. ^** P <0.01, ^*** <0.001.

Example  8: hBSMC  Transplantation is capillary and CMC  Increase density and reduce myocardial fibrosis Soshikim

To determine the final impact of hBMSC transplantation on the pathological characteristics of infarcted myocardium, capillary and CMC densities were quantified after CD31 and HE contamination and also measured% columnar fibrosis. Capillary density was 2 and 2.3 times higher in hBMSC transplanted rats than in TBMC and PBS injected rats (P <0.01) (FIG. 9 a-d). CMC density was also 2.4 and 2.8 times higher in hBMSC transplanted rats than in TBMC and PBS injected rats (P <0.01) (FIG. 9 d-g). The% circumferential fibrosis area, measured in the trichrome-contaminated portion of Masson, demonstrated smaller areas of fibrosis in the hBMSC transplanted heart (P <0.001 vs TBMC, PBS).

9A-G are described in more detail as follows. ad . Representative immunohistochemical discovery of CD31 (ac) in infarct myocardium 4 weeks after cell transplantation showing significantly higher capillary density in hBMSC transplanted heart. ^** P <0.01 vs TBMC and PBS. eh. Representative findings of infarcted areas contaminated with HE (eg) demonstrate the marked structure and regeneration of myocardium in the hBMSC transplanted heart. ^** <0.01 vs TBMC and PBS. Representative findings of maternal trichrome-contaminated heart showing a significantly smaller area of% fibrosis in the hBMSC transplanted heart. ^** <0.01 vs TBMC and PBS.

The following materials and methods were used as needed to perform the experiments described above in Examples 1-8.

1. Isolation and Culture of BMSC

New raw human BMs from young male donors were purchased from Biowhitttaker (Cambrex) (Wakersville, MD). BM was centrifuged at 1300 rpm for 10 minutes to obtain cell pellets. Cell pellets were resuspended in 25 ml of DPBS containing 0.5 M EDTA (DPBS-E). After centrifugation for 7 min at 1300 rpm, the cells were resuspended in 5 ml DPBS-E and 20 ml NH ₄ Cl for induction of hemolysis. After centrifugation and washing with DPBS-E, the cells were filtered through a 40-μm nylon filter and plated in 6-well plates coated with fibronectin (100 μg / ml). Cells were 37 in complete DMEM with low (1 g) glucose containing 17% of FBS (Biowhittaker; a lot selected to promote expansion of bone marrow cells), 100 U / ml phenylsilin, and 100 μg / ml streptomycin and 2 mM glutamate. Grows for 4-6 hours at &lt; RTI ID = 0.0 &gt; C &lt; / RTI &gt; and 5% CO ₂ . Medium was replaced with fresh complete medium. Adhesion cells were grown to 60% confluence. Cells were then grown in complete medium into 25 cm ² tissue culture flasks (T25) at a density of ¹ × 10 ⁴ cells / cm ² . After the cells were 60% confluent, the cells grew sequentially with T75 and T175 at the same density. After at least two generations of cultures in T175 flasks, cells were labeled with CellTracker ^™ CM-DiI (molecular probe) and plated into wells of 96-well plates at a density of 1/2 cell per well by limiting dilution, Cultures were collected with collected control medium before limiting dilution, stored at -80 ° C and filtered through a 0.2 μm filter. Under fluorescence microscopy, the inventors excluded that wells containing one or more cells (FIG. 1A) showed phase contrast and luminescent images of single cells in the wells. When cells grew to 40-50% confluent, cells from one well grew naturally in one well of a 6-well plate, then T25, T75, and at a density of 4-8 × 10 ³ cells / cm ² . It grew naturally in T175. Cells were then incubated in T175, plated at 1: 10-40 dilution again and grown to 4-8 × 10 ³ cells / cm ² .

2. Luminescence-Activated Cell Sorting

Luminescence-activated cell sorting (FACS) analysis of hBMSCs was performed on cultured hBMSCs. Cells were used from at least three different clones of pre- and post-clonal isolation and also two sets of cells at 5PDs and 120PDs for the clonal line. The procedure for FACS contamination was described previously Kalka, 2000 # 11. In sum, a total of 2 × 10 ⁵ cultured cells were resuspended in 200 μl of Dulbecco's PBS (Cambrex) containing 10% FBS and 0.01% NaN ₃ and directly PE- or FITC-binding monoclonal antibody or non-binding. Antibodies were then incubated for 20 minutes at 4 ° C. by FITC-binding rabbit anti-mouse IgF (Jackson Immunology Study). Appropriate isotype-identical immunoglobin served as a control (Beckton-Dickinson, Medford, Mass., USA). After contamination, cells were fixed in 2% paraformaldehyde. Quantitative FACS was performed on a FACStar flow cytometer (BD). Antibodies used for FACS analysis were CD4, CD8, CD11b (Mac-1), CD13, CD14, CD15, CD29, CD30, CD31, CD34, CD44, CD44, CD71, CD73, CVD90 (Thyl), CD117 (c-kit). ), CD146, CD166, HLA-DR, HLA-ABD, β2-microglobulin from Mcton Dickson (BD), CD133 (AC133) from Miltenyl Biotech (Auburn, CA, USA), and Ancell (Bayport, MN, USA) CD105 from Endoglin, and unbound antibody against Oct4 from Santa-Cruz. For comparison, human mesenchymal stem cells (PT-2501) and medium (PT-3001) were purchased from Cambrex (Poietics) and also used as FACS.

3. DNA ploidy analysis

DNA content per cell was measured by pre-treatment with ribonuclease (100 μg / ml) followed by contamination with prodium iodide (50 μg / ml) followed by FACS analysis.

4. Telomere Length Analysis

We used the TeloTAGGG telomere length analysis kit (Roche, Indianapolis, IN) for the measurement of the telomere length of hBMSCs at 5 PDs and 120 PDs from three different clones. In sum, after isolation (1 μg) and digestion of genomic DNA, DNA fractions were separated by gel electrophoresis and transferred to nylon membranes by Southern blotting. Blotted DNA fragments were crossed with a digoxigenin (DIG) -labeled probe specific for telomere restriction enzyme fragment (TRF) and then incubated with DIG-specific ab covalently bound with alkaline phosphate. Finally, immobilized telomeres probes are visualized by CDP-star, a highly sensitive chemiluminescent material. Average TRF length was determined by comparing signals against molecular weight standards.

5. In Vitro Differentiation of hBMSCs

All of the following ex vivo studies were performed using three different cronal hBMSCs with 5 and 90 PDs. To induce differentiation into ECs, hBMSCs were diluted with 0.1% gelatin or vitre in DMEM or EBM-2 (Clonectics) with 2-5% FBS, ^10-8 dexamethasone and 10 ng / ml VEGF (R & D, Nineapolis, MN). Plates were again incubated at 5 × 10 ⁴ cells / cm ² in nectin coated glass chamber slides and incubated for up to 14 days. To induce differentiation into the SMC phenotype, hBMSCs were re- plated (50ng / ml, R & D) in non-coated or fibronectane coated petri dishes in 1-2% DMEM or EBM-2 supplemented with PDGF-Bb [ Hellstrom, 1999 # 756; Yamashita, 2000 # 757], and incubated for 14 days. To induce neural passage differentiation, hBMSCs were combined with 100 ng / ml nFGF and 20 ng / ml EGF, B27 supplement in DMEM / F12, and 4 x 10 on poly-L-ornithine-laminin coated dishes (BD) or plastic dishes. Plate in ⁴ cells / cm ² [Palmer, 1999 # 764; Mezey, 2000 # 750; Brazelton, 2000 # 749], up to 14 days. To induce endothelial passage differentiation, hBMSCs were 3-5 × 10 ⁴ cells on 1% Matrigel (BD) in 2% FBS, 2ng / ml HGF, 10ng / ml FGF-4, supplemented with ^10-8 dexamethasone Plates were incubated at / cm ² and incubated for 7 days. Then 10 mg / ml DMSO or 0.5 mM sodium butyrate was added and incubated for at least 7 days [Shen, 2000 # 761; Hamazaki, 2001 # 762; Oh, 2000 # 763; Schwartz, 2002 # 747]. To induce CMC differentiation of hBMSCs, hBMSCs were cocultured with freshly isolated NRCRs. Primary NRCMs from F344 rats were isolated as described above and plated at 2 × 10 ⁵ cells / cm ² [De Luca, 2000 # 759]. DiI-labeled hBMSCs were added to NRCM culture plates at a ratio of 1: 4 in DMEM containing 10% fetal calf serum (FCS) and incubated for up to 10 days [Condorelli, 2001 # 758].

6. In Vitro Fusion Research

Rat aortic endothelial cells (RAECs) were cultured in EBM-2 containing EGM-2 MV Single Quots (Cambrex). Rat vascular flexible muscle cells (RVSMCs) were incubated in DMEM with 1 g / L glucose (Cellgro) containing 15% FBS, 2 mM L-glutamine and antibiotics. NRCMs were isolated and incubated as described above. When cells reached 50-60% confluence (within 3 days), cells were contaminated with 25 μM of Vybrant CFDA SE Cell Tracer Kit (Molecular Probe, Engene, OR) according to the manufacturer's instructions. Cells labeled with CFDA SE appear green under luminescence microscopy. After 2 days, DiI-labeled hBMSCs (red luminescence) were added to a culture plate of RAECs, RVSMCs or NRCM at a ratio of 1: 4 and incubated for 7 days. Medium changed every 2-4 days. To determine the preponderance of fusion and differentiation, 5000 cells were counted per co-culture from three different experiments.

7. Antibodies Used for Immunoluminescent Cytochemistry

For immunocytochemistry, cells are fixed in 4% cold paraformaldehyde for 7 minutes

Wash twice with PBS. As EC markers we found vWF (goat pAb) (1: 400, Sigma, St Louis, MO), Flk-1 (mouse mAb) (1: 300, Santa-Cruz, CA) VE-Cadherin (mouse mAb) ( 1: 100, BD), CD31 (mouse mAb) (1: 100, BD), UAE-1 lectin (1: 200, vector, Burlingame, CA), isolectin B4 (1: 200, vector) and DiI-acetyl Abs were used for the LDL (Biomedical Technologies, Stoughton, Mass.). As SMC markers we used abs for α-flexible muscle actin (mouse mAb) (1:30), calfonyl (mouse mAb) (1: 250, DAKO, Carpinteria, CA, USA). As neural passage markers, we found NF-200 (mouse mAb) (1: 400, Sigma), β-tubulin III (mouse mAb) (1: 100, Sigma), Gal-C (rabbit pAb) (1 Abs were used for: 100, Sigma) and GFAP (goat pAb) (1: 200, Santa-Cruz). As endoderm (hepatocyte or other endothelial) passage markers, we found CK18 (mouse mAb; 1: 300) (Sigma), α-fetoprotein (goat pAb, 1: 200) (Santa-Cruz), albumin (mouse mAb, 1: 400) (Sigma), HNF3β (goat pAb, 1: 100) (Santa-Cruz), HNF1α (Rabbit pAb, 1: 200) (Santa-Cruz) were used. As CMC markers, cTn I (two forms: mouse mAb and rabbit pAb, 1: 100) (Chemicon, Temecular, CA), ventricular myosin heavy chain α (α-MHC) (mouse mAb, 1: 100) (Chemicon), abs for α-sarcomeric actinine (clone EA-53, mouse mAb, 1: 200) (Sigma), ANP (rabbit pAb) (Chemicon) was used. Control mouse, rabbit or goat IgGs were prepared from Sigma. Secondary anti-mouse, rabbit or goat antibodies (AMCA, 1: 150, FITC or Cy-2, 1: 200; Cy-3, 1: 200) were prepared from Jackson Immunology (West Grove, PA).

8. Study Design of In Vivo Experiments

All procedures were performed on 6-7 week old female nude rats (Hsd: RH-rnu rats, Harlan, Indianapolis, IN) and immediately after generating acute MI, 5 sites in the peripheral infarct area (base front, center front, Intramyocardial injection of 8 × 10 ⁵ hBMSC or 8 × 10 ⁵ human press total BM cells (TBMC) or PBS at a total volume of 200 μl from the medial side, apical front, apical side) was performed. Rats were randomly divided into 15 groups each. Preparation of TBMCs was the same as preparation of hBMSCs before culture. hBMSCs and TBMCs were labeled with a carbocyanine dye, DiI (1.5 μl / ml, molecular probe) prior to cell transplantation as described above [Kalka, 2000; Kawamoto, 2001]. Separate rats (n = 4, each group) were small osmotic pumps injected with 1.25 mg BrdU / d (Sigma) (dissolved in a 1: 1 (vol / vol) mixture of dimethyl sulfoxide and 0.154 m NaCl) after surgery. Model 2ML4, Alzet, Palo Alto, Calif.) Was implanted under the back skin and sacrificed at 4 weeks [McEwan, 1998. TUENL]. For evaluation and molecular studies, another rat was similarly manipulated to sacrifice one and two weeks after surgery (n = 4, score each time). Following surgery, rats underwent echocardiography (Echo) and invasive hemodynamic measurements after 28 days. Rats were then killed by saline perfusion into the aorta. At necropsy, the heart was cut into three cross sections from apical to baseline, fixed with 4% paraformaldehyde, methanol or frozen in OCT compounds and divided into 5 μm thick.

9. Acute Myocardial Infarction Model

MI was induced as described above in the experiment [Kawamoto, 2003; Kawamoto, 2001]. After the right thoracic thoracic and incision of the pericadium under artificial gout (Model 683, Harvard's device), the left frontal descending (LAD) coronary artery was bound with 6-0 Proren sutures (Ethicon) at its origin.

10. Heart function measurement

Transthoracic echocardiography was performed with a 6.0-15.0 MHz ultraband linear transducer coupled to SONOS 5500 (Agilent Technologies, Andover, MA, USA) as described above. Kawamoto, 2003. LVEDD, LVESD, FS were measured. All measurements represent the average of at least three consecutive cardiac cycles. To assess regional wall motion abnormalities, a wall motion score index (WMSI) was used. OhJK, 1999. LV wall motion analysis is based on grading contractility of individual segments. Higher scores in this score system indicate more severe wall motion abnormalities (1, normal; 2, motor dysfunction, 3, motor dysfunction; 4, dyskinesia; 5, neurological dysfunction). EMSI is derived by dividing the sum of the wall cloud scores by the number of visualization segments; This indicates the extent of local wall cloud abnormalities. Normal WMSI is one. To measure hemodynamic parameters, a 1.4-French high-fitting pressure transducer (Micro-tip catheter, Millar Instrument, Houston, TX) was introduced into the right ventricle via the right carotid artery. After hemodynamics stabilized, LVSP, LVEDP, + dP / dt and -dP / dt were recorded using Polygraph (Model 9P) (Glass Instrument, West Warwick, RI, USA) [Kawamoto, 2001].

11. Immunoluminescent Histochemistry of Cardiac Tissue

OCT embedded frozen sections were fixed with 4% PFA and used for the next immunohistochemistry. For the identification of ECs, biotinylated isolectin B4 (1: 200; Vector) was used as primary Ans followed by streptavidin-FITC (1: 100) [Kawamoto, 2001]. SMCs were identified by mouse -SMC Ab (Sigma, 1: 200) followed by FITC-binding goat anti-mouse IgG. CMCs identified abs for cTnI and ANP followed by FITC-bound goat anti-rabbit IgG.

12. BrdU immunohistochemistry and measurement of apoptosis by TUNEL

BrdU was searched with sheep anti-BrdU antibody (1:50; Biodesign) followed by streptavidin-FITC (1: 100, vector). Nuclear contamination was performed with DAPI. BrdU positive cells were counted in the peri-infarct region where scar tissue contained less than 20% of the visible field (x 200 magnification).

In-reactor labeling of the fractionated DNA is performed by the terminal deoxynucleodidiltransferase (TdT) -mediated dUTP-biotinic end labeling (TUNEL) method by use of an in-cell cell search kit (Roche) as described above. [Fujio, 2000]. Two week myocardial sections were used for TUNEL contamination (n = 4, respectively). In short, the sections were treated with 20 μg proteinase K. After washing, the portions were counter-contaminated in a solution of fluorescein dUTP mixture and TdT for localization of the nucleus. To determine the proportion of aportocicle nuclei in CMCs or ECs, tissue was counter-contaminated with a mAb for -sarcomeric actinin or ILB4. Tissue sections were examined under a microscope at x 200 magnification. The total sum of irregular visible areas (total -10,000 nuclei) in the infarct / peripheral color area was examined. The percentage of apoptotic cells named the apoptotic index.

13. RT-PCT Analysis

RT-PCT was performed as described above Yoon, 2003. Total RNA was extracted from the culture cells or from the heart using a RNaqueous kit (Ambion, Austin, Texas, USA) according to the manufacturer's instructions. One microgram of total RNA was reversible transcription using random hexamer and Moloney murine leuchemia versus reversible transcriptase (Superscript II kit, Roche). RT products were processed by PCR using the Advantage cDNA Polymerase Mix (Clontech) or Taq Polymerase (Roche). For semiquantitative RT-PCT, quantification of mRNA expression of each gene was calculated based on GAPDH expression. PCR primers and annealing temperatures used are described in Table 1. RT_PCT products were analyzed by 1.5% agarose gel electrophoresis on 100-bp ladder (Life Echnologies) and quantified by UV image Eagle-Eye II (Stratagene).

14. Analysis of Cardiac Histology and Morphology

Capillary density and CMC density were counted after contaminating the 4 week sample with mAn for CD31 and H & K as described above Kawamoto, 2003; Kawamoto, 2001. For the modulus, the sum of the eight visible areas where the cross-sections of the capillaries and CMCs were clearly visible was chosen irregularly in the infarct / pericolor area and the number of capillaries or CMCs was x 200 magnification (n = 6, each). Group). To determine the fibrosis area, the average ratio of fibrosis area per LV area was measured for all histological sections after contamination with trichrome (Sigma) of Masson as described above (Kawamoto, 2000).

15. Statistical Analysis.

Statistical analysis was performed with two groups, ANOVA followed by unpaired student t-tests for comparison of Scheffe's post-hoc in two or more groups. p <0.05 is considered to indicate statistical criticality.

16. Polymerase Chain Reaction (PCR) Primer.

Table 1 below shows the oligonucleotide primers used as needed to perform the experiments described herein. “Product size” belongs to the expected size (reference pair) of the enlarged product.

Table 1. RT-PCT Primer Sequences and Expected Product Sizes

-f: front primer. -r: reversible primer

Supplementary Information

10. FACS analysis using multiple surface epitopes demonstrated zero to minimal expression (<1%) of CD117 in hBMSCs. Major histocompatibility complex (MHC) class I (ABC) and II (DR) were negative and no known HSC markers (CD34, CD133, Flk-1, Tie2) were expressed.

The following references 1-80 are mentioned throughout this specification and are also incorporated herein by reference.

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While the invention has been described with reference to specific embodiments, modifications and variations of the invention can be made without departing from the scope of the invention as defined in the following claims. All literature is incorporated herein by reference.

Claims (44)

Undetectable or low levels of the following cell markers as measured by standard cell marker detection assay: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, An isolated myeloid stem cell having at least one and preferably all of GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor. The isolated bone marrow stem cell of claim 1, wherein the cells are nearly spherical as measured by examination and have a diameter of less than about 25 to 35 micrometers, preferably less than about 15 micrometers. The isolated bone marrow stem cell of claim 1, wherein the cell has an average telomere restriction fragment length (TRF) of less than about 30 to 40 kilobases. The isolated myeloid stem cell according to any one of claims 1 to 3, wherein the cells are substantially euploid. 5. The isolated myeloid stem cell according to claim 1, wherein the forward ploidy of the cell is substantially maintained in cell culture for at least about 10 generations, preferably about 20 to about 200 generations. . The isolated bone marrow stem cell according to any one of claims 1 to 5, wherein the cells form endothelial cells (ECs) after contact with EC promoting conditions as measured by standard EC differentiation assays. 7. The isolated bone marrow stem cell of claim 6, wherein the EC promoting condition comprises contact with a vascular endothelial growth factor (VEGF). 8. The isolated bone marrow stem cell according to any one of claims 1 to 7, wherein the cells form smooth muscle cells (SMCs) after contact with SMC promoting conditions as measured by standard SMC differentiation assays. The isolated bone marrow stem cell of claim 8, wherein the SMC promoting condition comprises contact with platelet-derived growth factor (PDGF). 10. The isolated bone marrow stem cell of claim 1, wherein the cells form neurons after contact with neuronal facilitation conditions as measured by standard neuronal differentiation assays. 11. The isolated bone marrow stem cell of claim 10, wherein the neuronal promoting condition comprises contact with hepatocyte growth factor (HGF). The isolated bone marrow stem cell of claim 11, wherein the neuronal cell promoting condition comprises further contact with fibroblast growth factor-4 (FGF-4). 13. The isolated bone marrow stem cell of claim 12, wherein the neuronal cell promoting condition comprises further contact with DMSO or a pharmaceutically acceptable butyrate salt. The isolated bone marrow stem cell of claim 1, wherein the cells form cardiomyocytes after contact with incidental cardiomyocytes. 15. The isolated bone marrow stem cell of claim 14, wherein at least a portion of the formation is associated with cell fusion between the cell and accessory cardiomyocytes. The isolated bone marrow stem cell of claim 1, wherein the accessory cardiomyocytes are maintained in vitro, ex vivo or in vivo. A graft comprising at least one of the isolated bone marrow stem cells of claim 1. 18. The implant of claim 17, wherein the implant further comprises cardiomyocytes produced from isolated bone marrow stem cells. 19. The implant of claim 17 or 18, wherein the implant further comprises ECs produced by isolated bone marrow stem cells. 20. The implant of any one of claims 17-19, wherein the implant further comprises SMCs produced by isolated bone marrow stem cells. 21. The implant of any one of claims 17-20, wherein the implant further comprises cells from the recipient, wherein the recipient cells and bone marrow stem cells are mammals. The implant of claim 21, wherein the recipient cells and the isolated myeloid stem cells are homologous, autologous or heterologous. The implant of claim 17, wherein the implant is maintained in vivo or ex vivo. 19. The implant of claim 18, wherein the cardiomyocytes produced from bone marrow cells express a CMC specific protein (cTnI). The implant of claim 19, wherein the ECs produced from the bone marrow cells express ILB-4. The implant of claim 20, wherein the SMCs produced from bone marrow cells express α-SMA. 27. A cell culture, tissue or organ comprising the implant of any one of claims 17-26. A method comprising administering at least one of the isolated bone marrow cells of claims 1 to 16 and the implant of claims 17 to 26 to a mammal in need thereof, wherein said administration is performed to determine the severity of the disease in the mammal. A method of preventing, treating or alleviating the severity of a heart disease that is sufficient to prevent, treat or alleviate. The method of claim 28, wherein the method further comprises culturing the mammalian cell or implant for at least about 1 week. The method of claim 29, wherein said culture in a mammal is about 2 to 8 weeks. 31. The method of any one of claims 28-30, wherein the method further comprises administering to the mammal at least one angiogenic factor. 32. The method of any one of claims 28-31, wherein the method further comprises administering at least one nucleic acid encoding at least one angiogen factor or functional fragment thereof. 33. The method of any one of claims 28-32, wherein the method further comprises administering endothelial progenitor cells (EPCs) to the mammal. The method of claim 33, wherein the method further comprises separating the EPCs from the mammal and contacting the EPCs with at least one angiogen factor ex vivo. 35. The method of any one of claims 28-34, wherein the heart disease is one or more of congestive heart failure (CHF), ischemic cardiomyopathy, myocardial ischemia or infarction. 36. The method of any one of claims 28-35, wherein the method further comprises monitoring heart function in a mammal. The cardiac function of claim 36, wherein the monitored cardiac function is ultrasound cardiac, ventricular end cardiac dilation dimension (LVEDD), end-heart contraction dimension (LVESD), fractional shortening (FS), wall motion score index (WMSI) and LV heart At least one of shrinkage pressure (LVSP). Directions for isolation of the isolated myeloid stem cells of any one of claims 1 to 16 and optionally cells from mammals; Instructions for making, maintaining, and / or using the implant of any one of claims 17-26; A pharmaceutical product for preventing, treating or alleviating the severity of a heart disease comprising at least one of the cell medium, tissue or organ of claim 24 and optionally instructions for preparing the same. The pharmaceutical product of claim 38, wherein the product further comprises at least one angiogen factor or functional fragment thereof. 40. The pharmaceutical product of claim 38 or 39, wherein the product further comprises at least one nucleic acid encoding at least one angiogen factor or functional fragment thereof. Population of isolated bone marrow stem cells obtained by the following steps: a) collecting bone marrow cells from a mammal wherein the cells have a size of less than about 100 microns, preferably less than about 50 microns and more preferably about 40 microns or less, b) culturing (expanding) the cells collected in the medium under conditions of selecting adherent cells, c) selecting adherent cells and expanding them in semi-nfluency in the medium, d) diluting the cultured cells sequentially in a chamber having a conditioned medium, wherein the dilution is a dilution sufficient to form a density of less than about 1 cell per chamber for clonal separation of expanded cells, and e) incubating (expanding) each of the clonal isolates and selecting a chamber with expanded cells to create an isolated bone marrow cell population. 41. The method according to claim 40, wherein said step has explored levels of the following cell markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, A population of isolated bone marrow stem cells, further comprising collecting cells that do not express at least one of the CD117, CD133, MHC class I receptor and MHC class II receptor. 17. A method for producing the isolated bone marrow stem cells of any one of claims 1 to 16, comprising the following steps: a) collecting bone marrow cells from a mammal wherein the cells have a size of less than about 100 microns, preferably less than about 50 microns and more preferably about 40 microns or less, b) culturing (expanding) the collecting cells in the medium under conditions of selecting adherent cells, c) selecting adherent cells and expanding these cells in semi-confluency in the medium, d) diluting the cultured cells sequentially in a chamber having a conditioned medium, wherein the dilution is sufficient dilution to form a density of less than about 1 cell per chamber for clonal separation of expanded cells, and e) incubating (expanding) each of the clonal isolates and selecting a chamber with expanded cells to create an isolated bone marrow cell population. 44. The method of claim 43, wherein said step is navigable at the following cellular markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, A method for producing isolated bone marrow stem cells, further comprising the step of collecting cells that do not express at least one of the CD117, CD133, MHC class I receptor and MHC class II receptor.

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