MULTILINEAGE-INDUCIBLE CELLS AND USES THEREOF

Cross Reference to Related Applications

This application claim priority benefit of provisional US 60/971,448, which was filed September 1 1 , 2007.

This application relates to, but does not claim priority benefit of, US 10/544,021 , which was filed July 28, 2005 and is incorporated in its entirety herein by reference.

Federally-Sponsored Research or Development

The U.S. Government has certain rights in this invention as provided for by the terms of Research & Development Contract No. 7734.01 awarded by the Department of Veterans Affairs.

Field of the Invention

This disclosure relates to multilineage-inducible cells isolated from biological sources and methods of isolating, differentiating, and using these cells or the differentiated cells.

Background of the Invention

Stem cells are unspecialized cells that can self renew indefinitely and also differentiate into more mature cells with specialized functions. Stem cells offer unprecedented opportunities for treatment of debilitating diseases and a new way to explore fundamental questions of biology. Human embryonic stem cells have been shown to develop into multiple tissue types and to exhibit long-term self renewal in culture; however, the use of human embryonic stem cells is controversial, given the diverse views held in society about the moral and legal status of the early embryo. The controversy has prompted scientists to find meaningful post-natal substitutes for embryonic stem cells. It is now known that cells having at least some of the characteristics of embryonic stem cells are present in after-born individuals, even throughout adulthood. Often called "adult stem cells," these cells can self renew for extended periods of time, and

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can give rise to cells with specialized morphology and function. Until recently, it was believed that adult stem cells found in one tissue type would not be capable of generating the specialized cell types of another tissue type. Moreover, it was not thought that an adult stem cell could differentiate into a cell type derived from an embryonic germ layer other than the one from which the stem cell derived.

Although primitive cell subpopulations with the potential to differentiate toward various cell lineages have been isolated from post-natal sources, few of such cells can maintain a broad and multilineage differentiation capacity, resembling the plasticity of embryonic stem cells. Additional multipotent post-natal cells, particularly from human sources, are needed, as are new methods of reproducibly isolating such cells.

Summary of the Invention

Cell culture conditions to isolate multilineage-inducible cells (MIAMI cells) are disclosed herein. In certain embodiments, such culture conditions may include, for example, extracellular matrix substrate, oxygen tension, growth factors, vitamins A and/or C, essential fatty acids, cell density, co-culture of cells, or combinations thereof. The disclosed MIAMI cells have unique molecular profiles. For example, in one embodiment, MIAMI cells express any combination of (i) stage-specific embryonic antigen 4 (SSEA4); (ii) transcription factors Oct-4, Rex-1, Sox2, and/or Nanog; (iii) optionally, at least one of CD29, CD81, or CD90; (iv) optionally, at least one of CD 122, CD 164, hepatocyte growth factor receptor (c-Met), bone morphogenetic protein receptor, type IB (BMP-receptor 1 B), or neurotrophic tyrosine kinase receptor, type 3 (NTRK3). Optionally, the multilineage-inducible cells do not express one or more of CD34, CD36, CD45, CD49b, CD71, CDl 17, CD133, and HLA-DR. MIAMI cells (and single- cell-derived colonies thereof) can be maintained in vitro without detectable changes in their characteristic molecular profile. Such in vitro MIAMI cell populations have multi- germ layer differentiation potential, and can be differentiated into endothelial cells or cardiomyocytes.

Endothelial cells differentiated from MIAMI cells, as well as cells differentiated toward the endothelial lineage so no longer multipotent, are disclosed herein along with cell culture conditions to induce such differentiation. Differentiation-inducing agents that may be used are one or more of vascular endothelial growth factor (VEGF), basic

fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor- 1 (IGF-I), and hydrocortisone. Differentiated endothelial cells may express markers such as, for example, CD31 (platelet-endothelial cell adhesion molecule- 1), CD36 (the receptor binding collagen, modified fatty acids, and thrombospondin), or von Willebrand factor (vWF).

Cardiomyocytes differentiated from MIAMI cells, as well as cells differentiated toward the cardiomyoctye lineage so no longer multipotent, are disclosed herein along with cell culture conditions to induce such differentiation. Differentiation-inducing agents that may be used are one or more of epidermal growth factor (EGF), platelet- derived growth factor-dimer BB (PDGF-BB), 5-azacytidine, insulin, and transforming growth factor beta 1 (TGFβl). Cardiomyocytes may express markers such as, for example, myosin light chain-2 (MYL-2), myosin light chain-7 (MYL-7), cardiac troponin I, type 3 (TNNI3), or cardiac troponin T, type 2 (TNNT2). Transdifferentiation of at least some endothelial cells to cardiomyocytes in vivo is also possible, especially in situ in a heart.

MIAMI cells and differentiated MIAMI cells are useful for, among other things, the treatment of a subject afflicted by or suffering from many types of diseases, including, for example, cardiovascular disease (e.g., atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke), promoting angiogenesis or neovascularization, ameliorating the effects of angina or ischemia, and other types of regenerative medicine.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

Brief Description of the Drawings

FIGURE 1 shows proliferation of MIAMI cells as a function of oxygen tension. Fig. IA shows the proliferation of MIAMI cells plated in 60-mm dishes in expansion medium at 1,500 cells/cm ² and then allowed to proliferate in 1, 3, 5, 10, or 21% pθ ₂ for up to 15 days. Each point and bar represent the mean and SEM for triplicate determinations; **p < 0.01 ; ***p < 0.001 compared to 21% pθ ₂ . Fig. IB shows [ ³ H] -thymidine incorporation in MIAMI cells at 3 and 21% pθ ₂ . MIAMI cells were plated in 24-well

plates in expansion medium at 10,000 cells/cm ² and then allowed to proliferate in either 21 or 3% pθ ₂ for the time periods indicated. Subsequently, TCA-precipitable [ ³ H]- thymidine was measured as an index of DNA synthesis. The data shown are representative of three experiments. Results are expressed as mean ± SEM of quadruplicates. ***p < 0.001 compared to 21% pθ ₂ .

FIGURE 2 shows the expression of embryonic stem cells markers of MIAMI cells enhanced by low oxygen tension. Fig. 2A shows semiquantitative RT-PCR analysis utilized to evaluate markers characteristic of embryonic stem cells. MIAMI cells were plated in 60-mm dishes at 1 ,500 cells/cm ² and then grown in 3% and 21% pθ ₂ in expansion medium for three weeks. Left panel: Expression, detected during the logarithmic amplification phase, of the transcription factors Oct-4 (573 bp, 30 cycles), Rex- 1 (306 bp, 30 cycles), hTERT (272 bp, 25 cycles), and ELF-I α (235 bp, 23 cycles). Right panel: Determination of the same gene products in MIAMI cells grown at 3% or 21 % pθ ₂ in the presence of osteoblastic differentiation medium. Fig. 2B shows flow cytometry histograms with the immunophenotype of MIAMI cells grown at 3% or 21% pθ ₂ . MIAMI cells were plated in 60-mm dishes at 1 ,500 cells/cm ² and then grown in 3% and 21% pθ ₂ in expansion medium for fifteen days. MIAMI cells grown at 21% pθ ₂ were positive for SSEA4. The black area shows the profile of the negative control. The data shown are representative of those obtained in four different experiments. FIGURE 3 shows the analysis of telomerase activity in MIAMI cells by telo- meric repeat amplification protocol (TRAP). MIAMI cells were grown in the presence of expansion medium at 3% pθ ₂ for 15 days. Cell pellets were resuspended with CHAPS buffer. Fifty microliters of reaction mixture were incubated at room temperature for 30 min and then subjected to PCR cycles of 94°C for 30 sec, 59°C for 30 sec, and 71 ⁰ C for 1 min for 33 times. Twenty-five microliters of TRAP product were analyzed by electrophoresis in 0.5x Tris-borate-EDTA buffer on 12% polyacrylamide nondenaturing gels and visualized with ethidium bromide to stain DNA. Lane 1, positive control: a telomerase- immortalized human foreskin fibroblast (hTERT-BJl) cell line; lane 2, MIAMI cells; lane 3, negative control: hTERT-BJl heated at 85°C for 10 min; lane 4 negative control: MIAMI cells heated at 85 ⁰ C for 10 min; lane 5, positive control from the kit; lane 6, buffer only from kit.

FIGURE 4 shows the expression of endothelial markers during culture of MIAMI cells in an endothelial-induction medium. RNA was extracted from cells after 5 days, 10 days, or 21 days of culture using TRIzol reagent (Invitrogen, Rockville, MD). It was reverse transcribed into cDNA. Expression analysis of endothelial markers was quantified by qRT-PCR using the LightCycler apparatus and cDNA from treated and control cells. Specific primers were used to quantify expression of endothelial markers CD31 (accession NM_000442, Fig. 4A), CD36 (accession NM_000072, Fig. 4B), and von Willebrand factor (accession NM_000552, Fig. 4C). Target gene expression was normalized to the expression level of elongation factor- lα (EF- lα), and changes in gene expression were expressed as fold-induction compared to their levels at day 0. C = Control; V = VEGF only; GF = growth factor mix; EGM = EGM-2 medium.

FIGURE 5 shows the expression of cardiomyocyte markers during culture of

MIAMI cells in a cardiomyogenic medium. RNA was extracted from cells after they were cultured for three days, seven days, or 10 days using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNA. Expression analysis of endothelial markers was quantified by qRT-PCR using the LightCycler apparatus and cDNA from treated and control cells. Specific primers were used to quantify expression of cardiomyocyte markers MYL-2 (accession NM_000432, Fig. 5A), MYL-7 (accession NM_021223, Fig. 5B), cTnT (accession NM 000364, Fig. 5C), and cTnl (accession NM 000363, Fig. 5D). Target gene expression was normalized to the expression level of elongation factor- lα (EF- lα), and changes in gene expression were expressed as fold-induction compared to their levels at day 0.

FIGURE 6 shows quantification of cell death in the hippocampal CAl region. In an organotypic culture (i.e., a slice of mouse brain cultured in vitro), which is an art- recognized model for ischemia, the cells that do not survive deprivation of oxygen and glucose are detected by propidium iodide (PI) fluorescence staining. Neuronal cell death in the cultured brain slice was quantified after oxygen-glucose deprivation for 40 min (OGD), a brain slice injected with 2-3 μL culture medium (i.e., negative control) after oxygen-glucose deprivation for one hour (OGD + Medium), and a brain slice injected with 2-3 μL medium containing about 7000 MIAMI cells after oxygen-glucose deprivation for one hour (OGD + MIAMI-A).

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Description of Specific Embodiments of the Invention

I. Introduction

Disclosed herein are isolated, multilineage-inducible cells, which express at least one of hepatocyte growth factor receptor (c-Met), bone morphogenetic protein receptor, type IB (BMP-receptor IB), or neurotrophic tyrosine kinase receptor, type 3 (NTRK3). In one embodiment, the cells further express at least one of CD29, CD81, CD90, or stage-specific embryonic antigen 4 (SSEA4). The cells may be isolated from a wide range of biological sources, including amniotic fluid or membrane, placenta, Wharton's jelly, bone marrow, vertebral bodies, peripheral blood, umbilical cord blood, iliac crest aspirate, fat, cartilage, muscle, skin, bone, teeth, liver, brain, or mixtures thereof. In particular examples, the biological sample is bone marrow. The cells may be isolated from a mammal: such as a human or other primate, cow, sheep, pig, dog, or rodent (e.g., rat or mouse). In some examples, the mammal from which the cell is isolated is a postmortem subject. Also disclosed are isolated, multilineage-inducible cells, which express at least one of CD29, CD81, CD90, or stage-specific embryonic antigen 4 (SSEA4), and at least one of CDl 22, CDl 64, hepatocyte growth factor receptor (c-Met), bone morphogenetic protein receptor, type IB (BMP -receptor IB), neurotrophic tyrosine kinase receptor, type 3 (NTRK3), Oct-4, or Rex-1. In one embodiment, isolated CD29 ⁺ , CD81 ⁺ , CD90 ⁺ , CD122 ⁺ , and CD164 ⁺ multilineage-inducible cells are disclosed. Optionally, the multilineage-inducible cells do not express at least one of CD34, CD36, CD45, CD49b, CD71, CDl 17, CD133, HLA-DR, and any combination thereof.

Methods of isolating multilineage-inducible cells are also disclosed. The methods include culturing a cell population isolated from a biological sample, including amniotic fluid or membrane, placenta, Wharton's jelly, bone marrow, vertebral bodies, peripheral blood, umbilical cord blood, iliac crest aspirate, fat, cartilage, muscle, skin, bone, teeth, liver, brain, or mixtures thereof, under low-oxygen conditions (such as, in certain examples, less than about 3% oxygen) to produce adherent cells and non-adherent cells; removing the non-adherent cells; and expanding the adherent cells. In some methods the biological sample is from a post-natal subject, including, in some embodiments, from a human post-natal subject. In some methods, the adherent cells and the non-adherent cells are co-cultured for at least seven days, or the cells are placed in a

cell culture container, which includes an extracellular matrix (ECM) substrate, such as fibronectin, collagen, laminin, vitronectin, polylysine, heparan sulfate proteoglycans, entactin, or a combination thereof. Multilineage-inducible cells isolated by these methods are also contemplated. A method of inducing endothelial differentiation of multilineage-inducible cells by culturing the multilineage-inducible cells in an endothelial-induction medium for at least about 10 days (or at least about 14 or about 21 days) is also disclosed. Endothelial-induction medium may comprise any combination of VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), IGF-I (insulin-like growth factor- 1), hydrocortisone, or functional equivalents thereof. The endothelial-induction medium may be substantially free of serum and have a defined combination of growth factors. Endothelial cell differentiation may include the cell-surface expression or secretion of at least CD31 (platelet-endothelial cell adhesion molecule- 1), CD36 (the receptor binding collagen, modified fatty acids, and thrombo- spondin), or von Willebrand factor (vWF).

A method of inducing cardiomyogenic differentiation of multilineage-inducible cells by culturing the multilineage-inducible cells in a cardiomyogenic medium for at least about 4 to about 6 days (or at least about 7 or 10 days) is also disclosed. Cardiomyogenic medium may comprise any combination of epidermal growth factor (EGF), platelet-derived growth factor-dimer BB (PDGF-BB), 5-azacytidine, insulin, and transforming growth factor beta 1 (TGFβl), or functional equivalents thereof. Cardiomyo- cyte differentiation may include the expression of at least myosin light chain-2 (MYL- 2), myosin light chain-7 (MYL-7), cardiac troponin I, type 3 (TNNI3), or cardiac troponin T, type 2 (TNNT2). Also disclosed herein are methods of treating a disorder, such as, but not limited to, a cardiac or vascular disorder, promoting angiogenesis or neovascularization by administering a therapeutically effective amount of the multilineage-inducible cells or cells differentiated therefrom to a subject. Some methods further include inducing the cells to differentiate, for example, into cardiocytes or endothelial cells in vitro or in vivo. In some methods, the cells are introduced locally into a subject, or, in other examples, the cells introduced systemically into the subject. Specific cardiovascular disorders include atherosclerosis, cardiomyopathy (dilated or ischemic), congestive

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heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, etc. In some embodiments, scar tissue may be replaced, angiogenesis or neovascularization may be promoted, or the effects of angina or ischemia may be ameliorated. Damaged vasculature or necrotic myocardium may be repaired and regenerated.

Pharmaceutical compositions including disclosed multilineage-inducible cells in a pharmaceutically acceptable carrier are also disclosed herein.

Kits including a container containing a purified population of multilineage- inducible cells described herein are also disclosed.

II. Abbreviations βME β-mercaptoethanol

BMMNCs bone marrow mononuclear cells BMP-receptor IB bone morphogenetic protein receptor, type IB c-Met HGF receptor CAM cellular adhesion molecule CNTFR ciliary neurotrophic factor receptor DMEM Dulbecco's modified Eagle medium DMSO dimethyl sulfoxide ECM extracellular matrix EF- lα translation elongation factor- 1 alpha ES cell embryonic stem cell FACS fluorescence activated cell sorting FBS fetal bovine serum FN fibronectin HLA human leukocyte antigen (class I or class II) hTERT human telomerase reverse transcriptase MAPCs multipotent adult progenitor cells MSCs marrow stromal cells NTRK3 neurotrophic tyrosine kinase receptor, type 3 SSEA4 stage-specific embryonic antigen 4

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III. Terms

Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of common terms may be found in Alberts et al., Molecular Biology of the Cell Fifth Edition, published by Garland Science, (ISBN 0815341059); Kendrew et al. (eds.), Encyclopedia of Molecular Biology, published by Blackwell Science, 1998 (ISBN 08654262 IX); Lewin, Genes IX, published by Jones & Bartlett, 2007 (ISBN 0763740632); and Meyers (ed.), Molecular Biology and Biotechnology, published by Wiley- VCH, 1995 (ISBN 0471186341).

In order to facilitate understanding of the various embodiments of the invention, the following explanations of specific terms are provided:

Adherent: Connected to, associated with, or affixed to, a substrate. For example, a cell that adheres to a cell culture dish in vitro, and grows attached thereto is adherent. Typically, an adherent cell will not wash off a surface to which it is attached by gentle washing with a buffered saline solution. In some cases, enzymatic solutions (such as trypsin-EDTA) may be used to disrupt the attachment between an adherent cell and the surface to which it is attached. In other circumstances, adherent cells may be physically detached from a surface using a tool designed for such purposes, such as a cell scraper.

A non-adherent cell is one that is not stably connected to, associated with, or affixed to a substrate. Cells grown in suspension culture are examples of non-adherent cells.

Biological sample: Any sample that may be obtained directly or indirectly from a living or postmortem subject (such as, a recently deceased), including whole blood, plasma, serum, amniotic fluid or membrane, placenta, Wharton's jelly, bone marrow, vertebral bodies, iliac crest aspirate, umbilical cord blood, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, sputum, fluid from ulcers and/or other surface eruptions, blisters, abscesses, and/or extracts of tissues, cells, or organs (such as, fat, cartilage, muscle, skin, bone, teeth, liver, or brain). The biological sample may also be a laboratory research sample such as a cell culture super- natant. The sample is collected or obtained using methods well known to those skilled in the art.

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Bone morphogenetic protein receptor, type IB (BMP- receptor IB or BMPRlB): A member of a family of transmembrane serine/threonine kinases, which are receptors for members of the TGF-β superfamily. Human BMPRlB cDNA encodes a 502 amino acid polypeptide that contains a single transmembrane domain and an intracellular serine/threonine kinase domain. BMPRlB mRNA is about 6.5 kb and is expressed in several human tissues, with highest levels in prostate and brain. BMPRlB is also known as activin receptor-like kinase 6 (or ALK6). (See, for example, ten Dijke et al., Science, 264:101-104, 1994; Ide et al., Oncogene, 14:1377-1382, 1997; Ide et al., Cytogenet. Cell Genet., 81 :285-286, 1998). CD29: A 13O kD antigen expressed, for example, in leukocytes. CD29 is also known as the βl-integrin subunit, which associates with CD49a in VLA-I integrin. Alternate names for CD29 include Fibronectin Receptor, Beta Subunit (FNRβ), Very Late Activation Protein, Beta (VLA-β).

CD81: A 26 kD integral membrane protein, also known as TAPAl, which is expressed on many human cell types (including lymphocytes). CD81 is believed to associate with CD 19 and CD21 to form B-cell coreceptor. CD81 is a member of the transmembrane pore integral membrane protein family.

CD90: An 18 kD glycoprotein antigen expressed, for example, on CD34 ⁺ human prothymocytes, fibroblasts and brain cells and on mouse T cells. CD90 is also known as Thy-1. It belongs to immunoglobulin supergene family, and consists of a single immunoglobulin homology unit that is either intermediate between V and C or somewhat more similar to a V homology unit.

CD122: The 75 kD β-chain of the interleukin-2 receptor (also known as, IL- 2Rβ). This antigen is expressed, for example, on natural killer cells, resting T cells, and some B cells.

CD164: A protein antigen of about 80 to 90 kD, which is expressed, for example, in human CD34 ⁺ hematopoietic progenitor cells (Zannettino et al., Blood, 92:2613-2628, 1998). CD164 belongs to a heterogeneous group of secreted or membrane-associated proteins called sialomucins. Sialomucins are believed to have two opposing functions in vivo: first, as cytoprotective or antiadhesive agents and, second, as adhesion receptors.

Differentiation: A process whereby relatively unspecialized cells (for example, multilineage-inducible cells) acquire specialized structural and/or functional features characteristic of mature cells. Similarly, "differentiate" refers to this process. Typically, during differentiation, cellular structures are altered, gene expression is reprogrammed, tissue-specific proteins appear, and sternness is lost.

Essential fatty acids: Fatty acids that are not synthesized by the body and thus must be obtained through dietary intake. There are ω-3 fatty acids (e.g., α-linoleic acid, eicosapentaenoic acid, docosahexaenoic acid) and ω-6 fatty acids (e.g., linoleic acid, γ- linolenic acid, dihomo-γ-linolenic acid, arachidonic acid). Both are needed. The fϊrst- named ω-3 and ω-6 fatty acids are considered short-chain polyunsaturated fatty acids and the others are long-chain polyunsaturated fatty acids.

Expand: A process by which the number or amount of cells in a cell culture is increased due to cell division. Similarly, the terms "expansion" or "expanded" refers to this process. The terms "proliferate," "proliferation" or "proliferated" may be used interchangeably with the words "expand," "expansion", or "expanded." Typically, during an expansion phase, the cells do not differentiate to form mature cells.

Expression: A process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Thus, the term "expression" contemplates either or both gene expression, measured for example, by levels of RNA (such as, mRNA) in a cell, or protein expression. Methods of determining RNA levels are well known in the art, and include Northern blots, RT-PCR, RNAse protection, and others. Methods of determining protein expression are similarly well known, and include Western blots, functional assays, immunofluorescence, optical absorbance, microscopy (including electron microscopy) and others.

Extracellular matrix (ECM): A complex network of different combinations of collagens, proteoglycans (PG), hyaluronic acid, laminin, fibronectin , and many other glycoproteins. The ECM is a scaffold that fills extracellular spaces. In some instances, the ECM (or particular components thereof) can mediate cell-to-cell interactions, or play a functional role in mediating cellular proliferation or differentiation.

Proteoglycans may be modified by glycosaminoglycans (GAGs), which are long-chain compounds of repeated disaccharide units. The four main types of GAGs

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consist mainly of sulfated heparan sulfate/heparin, chondroitin sulfate/dermatan, keratan sulfate, and the non-sulfated glycosaminoglycan hyaluronic acid. Many proteoglycans contain a core protein which links them to the cellular membrane. Hyaluronic acid is the only extracellular oligosaccharide that is not known to be covalently linked to a protein.

Growth factor: Substance that promotes growth, survival, and/or differentiation of a cell. Growth factors include molecules that function as growth stimulators (mitogens), molecules that function as growth inhibitors (e.g., negative growth factors) factors that stimulate cell migration, factors that function as chemotactic agents or inhibit cell migration or invasion of tumor cells, factors that modulate differentiated functions of cells, factors involved in apoptosis, or factors that promote survival of cells without influencing growth and differentiation. Examples of growth factors are bFGF, EGF, CNTF, HGF, NGF, and activin-A.

Hepatocyte growth factor receptor (c-Met): Tyrosine kinase comprised of disulfide-linked subunits of about 50 kD (alpha) and about 145 kD (beta), which is a receptor for hepatocyte growth factor. In the fully processed c-Met product, the alpha subunit is extracellular, and the beta subunit has extracellular, transmembrane, and tyrosine kinase domains as well as sites of tyrosine phosphorylation. (See, for example, Bottaro et al, Science, 251 :802-804, 1991). Induce: To cause to move forward to a result. For example, certain cell culture conditions may establish an environment that prompts one or more events (sometime, a cascade of events), which results in the specialization of a previously unspecialized cell type. In this example, placing an unspecialized cell into such culture conditions induces the cell to become more specialized, such as to differentiate. Isolated: An "isolated" cell is a cell that has been purified from other cell types and components of a tissue. Cells can be isolated by a variety of methods, including mechanical and/or enzymatic methods. In one embodiment, an isolated population of cells includes greater than about 50%, greater than about 75%, greater than about 90%, greater than about 95%, or greater than about 99% of the cells of interest. In another embodiment, an isolated population of cells is one in which no other cells of a different phenotype can be detected. In a further embodiment, an isolate population of cells is a population of cells that includes less than about 5%, or less than about 1% of cells of a

different phenotype than the cells of interest. An "isolated" cell may be a population of clonally derived cells, such as cells expanded into a single-cell-derived colony.

Low density: A relatively small number of cells per unit area of a container in which the cells are contained. Adherent cells are often considered to be at low density when the population of cells do not form a continuous monolayer on the surface on which the cells are adhered (e.g., less than about 30% confluent). Exemplary cell densities include no more than about 10 ⁴ cells/cm ² , or no more than about 10 ⁵ cells/cm ² .

Low-oxygen tension (or low oxygen conditions): Any culturing conditions below the normal atmospheric oxygen level (which is about 21%). Thus, in particular embodiments, low oxygen conditions are less than about 15% oxygen, less than about 10% oxygen, less than about 5% oxygen, less than about 3% oxygen, more than about 2% oxygen, more than about 1% oxygen, or any range therebetween. In some embodiments, the culture oxygen conditions are kept as close as possible to the normal physiological oxygen conditions in which a particular cell would be found in in vivo. This may mean that the oxygen conditions employed for a particular cell type will depend on the regional origin of that particular cell type. For example, cells from an alveolar origin may prefer growth at about 14% oxygen, cells from an arterial source will prefer an oxygen concentration of about oxygen, whereas those from certain regions of the brain may prefer oxygen conditions as low as about 1.5% oxygen. Low oxygen condi- tions are not to be considered the same as "hypoxic" conditions. Low oxygen conditions are intended to mimic physiological conditions, whereas hypoxic conditions describe oxygen levels that are less than normal physiological conditions for a particular cell type.

Marker: A protein, glycoprotein, or other molecule expressed on the surface of a cell, which serves to help identify the cell. Markers can generally be detected by conventional methods. Specific, non-limiting examples of methods for detection of a cell surface marker are immunohistochemistry (e.g., epifluorescence microscopy), fluorescence activated cell analysis or sorting, or enzymatic analysis. Other markers that are not expressed on the surface of a cell, such as a transcription factor, can be detected by their RNA or protein expression. Specific, non-limiting examples of methods for detection of both types of markers are amplification with primers (e.g., PCR), hybridization with a probe (e.g., Northern or dot blotting), arrays of substrate-

attached probes (e.g., Affymetrix's GeneChip or Sequenom's MassARRAY systems), immunoprecipitation, ELISA, or Western blotting.

Multilineage-inducible cell: Cell capable of differentiating into more than one cell lineage. A multilineage cell is capable of differentiating into cell types derived from more than one germ layer, including cell types of mesodermal, ectodermal or endodermal origin. In particular examples, a multilineage-inducible cell can be differentiated into mesodermal, neuroectodermal, and endodermal cell lineages, including, for instance, osteoblasts, chondrocytes, adipocytes, neurons, and β-like cells.

Nanog: A developmentally regulated, mammalian transcription factor containing a homeo domain, which is characteristically expressed in undifferentiated pluripotent embryonic stem cells (see, for example, Mitsui et al, Cell, 1 13:631-642, 2003; Chambers et al., Cell, 1 13:643-655, 2003).

Neurotrophin-3 receptor (NTRK3) (also known as gpl45 or trkC): A member of the TRK family of tyrosine protein kinase genes, which is expressed, for example, regions of the brain, including the hippocampus, cerebral cortex, and the granular cell layer of the cerebellum. In one embodiment, NTRK3 is a glycoprotein of about 145 kD and a receptor for neurotrophin-3 (see, for example, Lamballe et al., Cell, 66:967-979, 1991 ; Valent et al, Eur. J. Hum. Genet., 5:102-104, 1997; McGregor et al, Genomics, 22:267-272, 1994). In other embodiments, there are four known splice variants in humans and two of the major variants are (i) full-length NTRK3 which contains an active intracellular PTK domain (gpl45-120 kDa) and (ii) truncated NTRK3 (TKd- NTRK3) which has a truncated inactive intracellular PTK domain (gp90 kDa) (Menn et al, J. Comp. Neurol, 401 :47-64, 1998; Menn et al, Eur. J. Neurosci. 12:3211-3223, 2000; Quartu et al, Dev. Neuro. 21 :309-320, 2003; Beltaifa et al, Eur. J. Neurosci. 21 :2433-2444, 2005) due to splice variance and a translational frameshift.

Oct-4 (also known as POU4F1): A developmentally regulated, mammalian transcription factor containing the POU homeo domain, which is characteristically expressed in undifferentiated pluripotent embryonic stem cells (see, for example, Flasza et al, Cloning Stem Cells, 5:339-354, 2003; Bhattacharya et al, Blood, 103:2956-2964, 2004; Sui et al, Differentiation, 71 :578-585, 2003; Palmieri et al, Dev. Biol, 166:259- 267, 1994).

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Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this invention may be conventional. Remington 's Pharmaceutical

Sciences, by E.W. Martin, Mack Publishing, Easton, PA, 17th Edition (1985), describes compositions and formulations suitable for pharmaceutical delivery of the multilineage- inducible cells herein disclosed.

In general, the nature of the carrier will depend on the particular mode and route of administration being employed for delivery of the mulilineage-inducible cells. For instance, parenteral formulations are usually injectable and comprise fluids that include pharmaceutically- and physiologically-acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Post-natal: After birth. For example, a neonate (a newborn), a child, an adolescent, or an adult (including, for example, an aged adult).

Rex-1 (also known as Zfp-42): A mammalian transcription factor containing zinc finger motifs, which is characteristically expressed in undifferentiated pluripotent embryonic stem cells (see, for example, Hosier et al., MoI Cell. Biol, 9:5623-5629,

1989; Rogers et al., Development, 113:815-824, 1991 ; Hosier et al, MoI Cell. Biol,

13:2919-2928, 1993; Nishiguchi et al, J. Biochem. (Tokyo), 116:128-139, 1994).

Sox2: A developmentally regulated, mammalian transcription factor containing a HMG-box DNA-binding domain, which is characteristically expressed in undifferentiated pluripotent embryonic stem cells (see, for example, Avilion et al, Genes Dev., 17:126-140, 2003; Sun et al, Crit. Rev. Eukaryot. Gene Expr., 16:211-231, 2006).

Stage-specific embryonic antigen 4 (SSEA4): A globoseries glycolipid

(related to SSEAl and SSEA3) recognized by monoclonal antibodies originally raised to distinguish early stages of mouse development. Primate pluripotent cells express

SSEA4 and SSEA3, while SSEAl is expressed only upon differentiation of such cells.

(See, for example, Andrews et al, Intl. J. Cancer, 66:806-816, 1996; Thomson &

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Marshall, Curr. Topics Dev. Biol., 38:133-165, 1998; Thomson et al., Science, 282: 1 145-1 147, 1998).

Subject: Any living or postmortem mammal, such as humans, non-human primates, cows, sheep, pigs, rodents (e.g., rats or mice) and the like which is to be the recipient of the particular treatment. In one embodiment, a subject is a human subject or a murine subject. Death of a subject is determined by any standard known in the art, including, for example, cessation of heart or brain function. A postmortem subject typically will have been dead for less than 48 hours, such as less than 24 hours. A postmortem subject may be housed in an environment that may slow cellular degrada- tion, which occurs following death; for example, a cool environment (such as between about 0°C and about 15°C, or between about 0 ⁰ C and about 10°C between about 0°C and about 4°C) may slow cellular degradation.

Therapeutically effective amount of a cell: An amount of a MIAMI cell (or a differentiated MIAMI cell) that can be determined by various methods, including gene- rating an empirical dose-response curve, potency and efficacy modeling, and other methods used in the biological sciences. In general, a therapeutically effective amount of MIAMI cells (or differentiated MIAMI cells) is an amount sufficient to alleviate at least one symptom of a disease or disorder to be treated in a subject. In one embodiment, a therapeutically effective amount of MIAMI cells (or differentiated MIAMI cells) is more than about 10,000 cells, more than about 20,000 cells, more than about 30,000 cells, or between about 5,000 cells and about 50,000 cells. In regenerative medicine, damaged or degenerating tissue of a subject is repaired by replacement with MIAMI cells that are transplanted and then differentiated in situ, or differentiated in vitro and then transplanted; alternatively, MIAMI cells or differentiated MIAMI cells may be transplanted and they then promote the division and/or differentiation of stem cells already resident within the tissue.

The therapeutically effective amount of cells will be dependent on the subject being treated (for example, the species or size of the subject), the degree that the subject is compromised, and the method and/or location of administration of the cells. In one embodiment, a therapeutically effective amount of cells is an amount of cells sufficient to measure MIAMI cells in the peripheral blood of a recipient.

Transduced and Transformed: A virus or vector "transduces" a cell when it transfers nucleic acid into the cell. A cell is "transformed" by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electropora- tion, lipofection, and particle gun acceleration.

Transplantation: Transfer of a tissue or an organ, or cells, from one body or part of the body to another body or part of the body. A "heterologous" transplantation occurs from one individual (donor) to another individual (recipient), wherein the individuals have genes at one or more loci that are not identical in the two individuals. Allogeneic transplantation occurs between individuals of the same species, who differ genetically. Xenogeneic transplantation occurs between individuals of two different species. An "autologous" transplantation is transplantation of a tissue or cells from one location to another in the same individual, or transplantation of a tissue or cells from one individual to another, wherein the two individuals are genetically identical.

Treating a disease: Refers to therapeutically intervening (or preventing) the partial or full development or progression of a disease, for example in a subject who is known to have a predisposition to a disease. An example of a subject with a known predisposition is someone with a family history of atherosclerosis or cardiomyopathy, or who has been exposed to environmental factors that predispose the subject to such disorders. Moreover, treating a disease refers to a therapeutic intervention that ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means including A or B, or including A and

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B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All patents, patent applications, books, and other publications mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Except as otherwise noted, the methods and techniques of the present invention are generally performed according to methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook & Russell, Molecular Cloning 3rd Ed., CSHL Press, 2001 ; Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, 2007; and Harlow & Lane, Using Antibodies, CSHL Press, 1999.

IV. MIAMI Cells

Disclosed herein are isolated post-natal, multilineage-inducible cells (MIAMI cells), which express a unique set of molecular markers. A summary of selected markers expressed (or not expressed, as applicable) in various MIAMI cell embodiments is shown in Table 1.

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TABLE 1. Representative MIAMI Cell Markers

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MIAMI cells may be identified by any unique set of the markers set forth in Table 1. For example, MIAMI cells may uniquely express at least one, at least two, at least three, at least four, at least five, or at least six of the Table 1 markers. In some embodiments, MIAMI cells express at least one of CD29, CD81, CD90, or stage- specific embryonic antigen 4 (SSEA4), and at least one of CD122, CD164, hepatocyte growth factor receptor (c-Met), bone morphogenetic protein receptor, type IB (BMP- receptor IB), neurotrophic tyrosine kinase receptor, type 3 (NTRK3), Oct-4, Rex-1, Sox2, or Nanog. In preferred embodiments, MIAMI cells express SSEA4, Oct-4, Rex- 1, Sox2, and Nanog. In other examples, MIAMI cells express at least one of c-Met, BMP-receptor IB, or NRTK3. In still other examples, MIAMI cells express at least a combination of CD29, CD81 , CD90, CD 122, and CD 164.

MIAMI cells are generally small cells. In some examples, they are between about 5 μm and about 12 μm, such as between about 7 μm and about 10 μm. MIAMI cells typically contain relatively little cytoplasm and are highly proliferative. In some examples, MIAMI cells have a population doubling time of about 20 hours to about 36 hours.

MIAMI cells can be isolated from a living or postmortem mammal of any age.

A mammal includes either human or non-human mammals. In some examples, MIAMI cells are isolated from post-natal subjects, such as a neonate, a child, an adolescent, or

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an adult (including, without limitation, an aged adult) of any mammalian species (e.g., primates, cows, sheep, pigs, dogs, rodents such as rats or mice). In particular embodiments, MIAMI cells are isolated from an adult primate, such as a human.

A subject from whom a biological sample is collected may be a postmortem subject. In particular examples, a postmortem subject is recently deceased. Death of a subject may be determined by any standard known in the art, such as cessation of heart function, or cessation of brain function. In some embodiments, a postmortem subject is one whose heart has recently (such as within about 1 hour to about 4 hours) stopped beating, or a subject who has no measurable brain activity, or a subject intended for organ or tissue donation. In other examples, a postmortem subject may have been dead for up to 48 hours, such as up to 24 hours or up to 12 hours. In particular examples, a postmortem subject may be housed in a cold environment, such as between about 0° to about 15°C, between about 1°C to about 10°C, or between about I ⁰ C to about 5°C, prior to collection of a biological sample. MIAMI cells may be isolated from one or more biological sample(s), such as amniotic fluid or membrane, placenta, Wharton's jelly, bone marrow, vertebral bodies, peripheral blood, umbilical cord blood, iliac crest aspirate, fat, cartilage, muscle, skin, bone, teeth, liver, or brain of a mammal. Methods for collection of a biological sample will vary depending upon, for example, the type of sample to be collected. Such collec- tion methods are well known in the art. For instance, bone marrow may be collected by inserting a needle into the marrow cavity of a bone under local anesthetic and aspirating marrow from the bone. Multilineage-inducible cells may be mobilized into peripheral blood by administering one or more growth factors (e.g., G-CSF) to the mammal. It may be useful (though not required) to maintain aseptic conditions during collection of a sample to reduce the possibility of bacterial, fungal, viral, or other infection in cell culture of MIAMI cells (see below).

MIAMI cells may be isolated from a biological sample by any method known in the art or a combination thereof, including without limitation the methods disclosed herein. In one embodiment, MIAMI cells are selectively expanded using cell culture techniques. In other embodiments, MIAMI cells are isolated from a biological sample based on the physical properties of the MIAMI cells. For example, several techniques are known in the art by which MIAMI cells may be isolated based on the unique set of

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markers expressed by MIAMI cells, including, for example, fluorescence-activated cell sorting (FACS), immobilized marker-specific antibodies (such as cell affinity methods like panning), or magnetic-activated cell sorting (MACS).

MIAMI cells may also be isolated on the basis of other physical properties of the MIAMI cells, such as cell size. A biological sample can be sorted on the basis of cell size using any method known in the art. For example, cells in a biological sample may be passed through one or more filters of varying pore size, including filters having a larger pore size, such as of about 50-200 μm, or about 80-100 μm, or filters having a smaller pore size, such as of about 10-50 μm, or 20-40 μm. In some examples, sequen- tial filters having decreasing pore size may be employed. In one embodiment, the cells passed through one or more filters are less than 40 μm in diameter. In other embodiments, isolated cells are between about 5 μm and 12 μm in diameter. The cellular component of a biological sample can also be sorted by size by passing a cell population through one or more size-exclusion column(s). In one such embodiment, the cells are. eluted along a size gradient such that the largest cells are eluted first and the smallest cells are eluted last. The cells can also be sorted by size using FACS. MIAMI cells may comprise more than about 10%, about 25%, about 50%, about 90% of size-sorted cell sample.

V. Methods of Isolating and Expanding MIAMI Cells

Methods for isolation and expansion of MIAMI cells have been identified and are disclosed herein. A method of isolation of the MIAMI cells includes obtaining a cell population from one or more biological sample(s), such as amniotic fluid or membrane, placenta, Wharton's jelly, bone marrow, vertebral bodies, peripheral blood, umbilical cord blood, iliac crest aspirate, fat, cartilage, muscle, skin, bone, teeth, liver, or brain. Methods for collecting a biological sample useful in the disclosed methods are the same as discussed above.

A biological sample, optionally, may be partially purified after collection. For example, non-cellular materials or dead or damaged cells may be removed by any technique known in the art. Though not bound by theory, it is believed that MIAMI cells respond favorably to signals (whether humoral, physical, or other signals) produced by one or more other cell types that reside with MIAMI cells within a

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biological compartment (from which a biological sample is taken); thus, isolation of MIAMI cells may be enhanced by maintaining MIAMI cells and such other resident cell type(s) in proximity to one another and/or in functional contact. In some embodiments, unfractionated cellular components of a biological sample will be retained for subsequent isolation of MIAMI cells. In other embodiments, a biological sample may be fractionated so as to co-fractionate MIAMI cells and any other cell types functionally relevant to MIAMI cells viability. In yet another embodiment, a biological sample may be fractionated to selectively remove cells (or other components) that do not reside with MIAMI cells, but which may be inadvertently included in a sample as a result of a particular cell collection process, such as connective tissue, or mature red blood cells.

In one specific, non-limiting example, the population of cells includes MIAMI cells as greater than 50% of the population, greater than 80% of the population, greater than 90% of the population, or greater than 95% of the population.

1. Cell Culture Methods

Once a biological sample from a mammal is collected and, as applicable, prepared, the cell population from the sample is further selected and expanded in culture medium. A cell population comprising at least one MIAMI cell is contemplated. In some examples, between about 10 ⁴ cells/cm ² and about 10 ⁶ cells/cm ² are used to seed the culture. In a particular example, about 10 ⁵ cells/cm ² are used to seed the culture.

The mean pθ ₂ in various tissues has been estimated from 5 to 71 torr (i.e., about 0.7% to about 9% oxygen at sea level) (Kaufman & Mitchell, Comp. Biochem. Physiol. A, 107: 673-678, 1994 ; Vollmar et al., Anesth. Analg., 75:421-430, 1992; Buerk & Nair, J. Appl. Physiol., 74:1723-1728, 1993 ; Levy et al., Pflugers Archiv., 407:388-95, 1986; Jiang et al., J. Appl. Physiol. 80:552-558, 1996). In comparison, pθ ₂ of tissue culture performed in room air is about 149 torr or about 21% oxygen (at sea level). A cell population containing MIAMI cells is cultured under low oxygen conditions. In some embodiments, low oxygen conditions comprise about 0.5% to about 10% oxygen, such as about 1% to about 5% oxygen, or about 1% to about 3% oxygen. In a particular example, low oxygen conditions comprise about 3% oxygen.

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In some methods, a subpopulation of cells from the sample adheres to a solid substrate (referred to as "adherent cells"), such as a cell culture container (for example, a culture dish, a culture flask, or beads designed for tissue culture). In some embodiments the solid substrate comprises an extracellular matrix (ECM) substrate. ECM substrates include, for example, fibronectin, collagen, laminin, vitronectin, polylysine, tenascin, elastin, proteoglycans (such as, heparan sulfate proteoglycans), entactin, Matrigel™, synthetic RGDS-containing peptides covalently crosslinked to hydrophobic biocompatible scaffolds (such as polyethylene glycol (PEG), polyglycolic acid (PGA), . poly(D,L-lactide-co-glycolide) (PLGA), or others), or a combination thereof. Any or all forms of a particular ECM substrate are contemplated herein. For example, collagen is commonly known to occur in multiple isoforms (Molecular Biology of the Cell, 3rd Ed., by Alberts et al, New York: Garland Publishing, 1994, Ch. 19), including eighteen different collagen isoforms (such as collagen I, II, III, IV, V, and others). Similarly, multiple isoforms of laminin (Ekblom et al, Ann. N. Y. Acad. ScL, 857:194-21 1, 1998) and fibronectin (Molecular Biology of the Cell, 3rd Ed., by Alberts et al., New York: Garland Publishing, 1994, Ch. 19) are known. In a specific, non-limiting embodiment, an ECM substrate comprises a 1-1000 ng/ml fibronectin-coated solid substrate, for example a 10 ng/ml fibronectin-coated solid substrate.

In other methods, adherent cells are co-cultured with cells from the biological sample, which do not adhere to a solid substrate and remain in suspension (i.e., "nonadherent cells"). Adherent and non-adherent cells may be co-cultured for various durations, such as for no less than about 3 days, no less than about 5 days, no less than about seven days, or no less than about 14 days. In a particular example, adherent and non-adherent cells are co-cultured for about 14 days. After which time, non-adherent cells may be removed from the culture.

The culture medium can be any medium or any buffer that maintains the viability of cells for at least maintaining them as multipotent, at least inducing them to differentiate, or at least maintaining them as differentiated cells. Numerous culture media are known and are suitable for use. The medium is generally prepared starting from a minimal essential medium such as low-glucose Dulbecco's modified Eagle medium (DMEM).

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The growth medium may be supplemented with serum containing growth factors. Specific, non-limiting examples of serum are horse, calf, or fetal bovine serum (FBS). The medium can have between about 2% by volume to about 10% by volume serum, or about 5% by volume serum, or about 2%. In one embodiment, a growth medium is supplemented with about 5% FBS. The growth medium may also be supplemented by media conditioned by culturing of non-adherent cells or adherent feeder cells.

The growth medium may also be supplemented with one or more vitamins. Specific, non-limiting examples of vitamins are ascorbic acid (vitamin C) and toco- pherol (vitamin E). The medium can have from about 1 μM to about 10 mM vitamin C, from about 10 μM to about 1 mM vitamin C, from about 20 μM to about 500 μM vitamin C, or about 100 μM vitamin C. The medium can have from about 3 nM to about 30 μM vitamin E, from about 30 nM to about 3 μM vitamin E, from about 150 nM to about 600 nM vitamin E, or about 300 nM vitamin E. In a particular example, the medium contains about 100 μM vitamin C and about 290 nM vitamin E.

The growth medium may also be supplemented with one or more fatty acids or sterols. Specific, non-limiting examples of fatty acids or sterols are linoleic acid, lipoic acid, arachidonic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmito- leic acid, stearic acid, cholesterol, and substituted cholesterols. The medium can have from about 0.1 nM to about 20 μM each of one or more fatty acids, about 1 nM to about 2 μM each of one or more fatty acids, or about 5 nM to about 250 nM each of one or more fatty acids. It can have from about 10 nM to about 100 μM of cholesterol or substituted cholesterols, about 100 nM to about 10 μM of cholesterol or substituted cholesterols, or about 200 nM to about 5 nM of cholesterol or substituted cholesterols. In a particular example, the medium contains about 200 nM linoleic acid, about 400 nM lipoic acid, about 10 nM arachidonic acid, about 1 μM cholesterol, about 100 nM linolenic acid, about 100 nM myristic acid, about 100 nM oleic acid, about 100 nM palmitic acid, about 100 nM palmitoleic acid, about 100 nM stearic acid, and about 1 μM cholesterol Specific growth factors and other differentiation-inducing agents may be added to the medium. Specific non-limiting examples of such additives include VEGF (from about 0.5 ng/ml to about 5 μg/ml, or from about 5 ng/ml to about 500 ng/ml), bFGF

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(from about 0.1 ng/ml to about 1 μg/ml, or from about 1 ng/ml to about 100 ng/ml), EGF (from about 0.1 ng/ml to about 1 μg/ml, or from about 1 ng/ml to about 100 ng/ml), IGF-I (from about 0.1 ng/ml to about 1 μg/ml, or from about 1 ng/ml to about 100 ng/ml), insulin (from about 0.01 nM to about 0.1 μM, or from about 0.1 nM to about 10 nM), PDGF-BB (from about 0.1 ng/ml to about 1 μg/ml, or from about 1 ng/ml to about 100 ng/ml), TGFβl (from about 0.1 ng/ml to about 1 μg/ml, or from about 1 ng/ml to about 100 ng/ml), hydrocortisone (from about 1 nM to about 10 μM, or from about 10 nM to about 1 μM), and 5-azacytosine (from about 10 nM to about 500 μM, or from about 0.5 μM to about 30 μM). In a non-limiting example, an endothelial-induction medium comprises about 50 ng/ml VEGF, about 10 ng/ml bFGF, about 10 ng/ml EGF, about 10 ng/ml IGF-I, and about 100 nM hydrocortisone; in another non-limiting example, a cardiomyogenic medium comprises about 2% FCS, about 10 ng/ml EGF, about 10 ng/ml PDGF-BB, about 3 μM 5-azacytidine, about 1 nM insulin, and about 10 ng/ml TGFβl. The medium may also contain one or more additional additives, such as antibiotics or nutrients. Specific non-limiting examples of antibiotics include 10- 1000 U/ml penicillin and about 0.01 mg/ml to about 10 mg/ml streptomycin. In a particular example, a growth medium contains about 100 U/ml penicillin and about 1 mg/ml streptomycin. In one embodiment, the cells are cultured in the growth medium for about seven days to about 20 days. In another embodiment, the cells are cultured in the growth medium for about 12 days to about 16 days. In a particular embodiment, the cells are cultured in the growth medium for about 14 days. Thereafter, single-cell-derived colonies of MIAMI cells may be isolated for expansion using any technique known in the art, such as cloning rings. Alternatively, single-cell-derived colonies of MIAMI cells may be pooled for expansion.

MIAMI cells are expanded under low oxygen conditions and in a growth medium as described above. In a particular example, MIAMI cells are expanded in growth medium supplemented with about 2% FBS. In another example, MIAMI cells are expanded in growth medium supplemented with about 100 U/ml penicillin and about 1 mg/ml streptomycin. In some embodiments, MIAMI cells are expanded on a

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solid substrate comprising an ECM substrate, such as (1-1000 ng/ml) fibronectin- coated substrates, for example a 10 ng/ml fibronectin-coated substrate.

2. Other Methods of MIAMI Cell Separation As unique sets of MIAMI cell markers have been disclosed herein, fluorescence activated cell sorting (FACS) may be one exemplary technique useful for isolating MIAMI cells. FACS can be used to sort cells that express a particular cell surface marker or set of cell surface markers by contacting the cells with one or more appropriately labeled antibody(ies). FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells. Any FACS technique may be employed as long as it is not detrimental to the viability of the desired cells. (For exemplary methods of FACS, see U.S. Patent 5,061 ,620). In one embodiment, multiple antibodies and FACS sorting can be used to produce isolated populations of CD29 ⁺ , CD81 ⁺ , CD90 ⁺ , CD122 ⁺ , CD164 ⁺ , multilineage-inducible cells, or to purify cells that express CD29, CD81 , CD90, CD122, and CD164, but do not express at least one of CDl 3, CD49b, or CD71. In other embodiments, FACS sorting can be used to produce isolated populations of multilineage-inducible cells that express at least one of CD29, CD81 , CD90, or stage-specific embryonic antigen 4 (SSEA4), and at least one of CD 122, CD 164, hepatocyte growth factor receptor (c-Met), bone morphogenetic protein receptor, type IB (BMP -receptor IB), neurotrophic tyrosine kinase receptor, type 3 (NTRK3), Oct-4, or Rex-1. In still other embodiments, post-natal, multilineage- inducible cells that express at least one of c-Met, BMP-receptor IB, or NTRK3 may be isolated by FACS. Other techniques of differing efficacy may be employed to purify and isolate desired populations of cells. The separation techniques employed should maximize the retention of viability of the fraction of the cells to be collected. The particular technique employed will, of course, depend upon the efficiency of separation, cytotoxicity of the method, the ease and speed of separation, and what equipment and/or technical skill is required.

Separation procedures may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents, either joined to a mono-

clonal antibody or used in conjunction with complement, and "panning," which utilizes a monoclonal antibody attached to a solid matrix, or another convenient technique. Antibodies attached to magnetic beads and other solid matrices, such as agarose beads, polystyrene beads, hollow fiber membranes and plastic petri dishes, allow for direct separation. Cells that are bound by the antibody can be removed from the cell suspension by simply physically separating the solid support from the cell suspension. The exact conditions and duration of incubation of the cells with the solid phase-linked antibodies will depend upon several factors specific to the system employed. The selection of appropriate conditions, however, is well within the skill in the art. The unbound cells then can be eluted or washed away with physiologic buffer after sufficient time has been allowed for the cells expressing a marker of interest (for example, CD29, CD81, CD90, CD 122, or CD 164) to bind to the solid-phase linked antibodies. The bound cells are then separated from the solid phase by any appropriate method, depending mainly upon the nature of the solid phase and the antibody employed.

Antibodies may be conjugated to biotin, which then can be removed with avidin or streptavidin bound to a support, or fluorochromes, which can be used with a fluorescence activated cell sorter (FACS), to enable cell separation (see above).

In one embodiment, MIAMI cells may be separated from other cells by the cell- surface expression of CD29, CD81, CD90, CD122, and CD164. In one specific, non- limiting example, CD29 ⁺ cells are positively selected by magnetic bead separation, wherein magnetic beads are coated with CD29-reactive antibody. The CD29 ⁺ cells are then removed from the magnetic beads, for example, by culture release or other methods known in the art. The CD29 ⁺ cells may be further purified, for example, in serial steps using magnetic beads coated with CD81-, then CD90-, then CDl 22-, and finally CD164-reactive antibody (as described for anti-CD29-coated beads). Alternatively, at any stage, different purification methods may be performed, such as FACS sorting the population of cells released from the magnetic beads.

In one embodiment, magnetic bead separation is used to first separate a popula- tion of cells that do not express at least one of CD34, CD36, CD49b, CD71, or CDl 33. In this embodiment, the unbound cells will be enriched for MIAMI cells. MIAMI cells may be separated from the enriched cell population as previously described. In addi-

tion, panning can be used to separate cells that do not express one or more specific markers, such as CD34, CD36, CD49b, CD71, or CD133 (see Small et al., J. Immunol. Metk, 167:103-107, 1994 for panning methods).

MIAMI cells isolated by these or other methods can be maintained in culture, such as described herein.

VI. Methods of Using Multilineage- Inducible Cells

Methods are provided for treating a subject suffering from a disease or disorder, such as cardiovascular disease, or alleviating the symptoms thereof, by administering MIAMI cells isolated, grown, and/or differentiated according to the methods disclosed. The cells can be administered alone or in conjunction with another pharmaceutical agent, such as a growth factor or immunosuppressive agent.

In one embodiment, MIAMI cells are isolated and a therapeutically effective amount of MIAMI cells is administered to the subject. In another embodiment, MIAMI cells are isolated and differentiated into a cell type useful for the desired treatment, for example into endothelial cells or cardiomyocytes, and a therapeutically effective amount of differentiated cells are administered to a subject, such as a human.

The cells may be administered in any fashion, for example in a dose of, for example about 10 ⁵ to about 10 ⁸ cells, such as about 10 ⁶ cells. Different dosages can of course be used depending on the clinical circumstances. For example, an aged subject (> 40 years old) whose symptoms include a reduced number of multilineage-inducible cells (e.g., less than about 20 per 10 ⁶ whole bone marrow cells plated) may be treated by administering MIAMI cells in an escalating series of doses. The cells may be administered systemically or locally. In another example, the cells can be administered in a gel matrix (such as Gelfoam from Pharmacia & Upjohn) which polymerizes to form a substrate in which the administered cells can grow.

In one embodiment, MIAMI cells or differentiated cells are administered systemically by injection of an aseptic, pyrogen-free composition. Specific, non-limiting examples include administration by intravenous injection, intramuscular injection, or subcutaneous injection. If administration is intravenous, a liquid suspension of cells can be prepared in injectible form and administered by a continuous drip or as a bolus. Any body cavity or blood vessel may be accessed by a cannula with an attached trocar.

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In another embodiment, MIAMI cells or differentiated cells are administered locally as an aseptic, pyrogen-free composition. Specific, non-limiting examples of local routes of administration include implantation and injection. The site of local administration will depend upon the particular disorder being treated. In some embodi- ments, a liquid suspension of cells can be prepared in injectible form or in a biocompatible medium which is injectible in liquid form and becomes semi-solid at the desired site. A conventional syringe or a controllable endoscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter (for example, 30 gauge or larger) that shear forces will not damage the MIAMI cells. The heart or the vasculature can be accessed with a catheter and it can be localized with radiography. Co-administering a contrast dye or radionuclide labeling allows visualization of where the cells are transplanted. After myocardial infarction, cells may be injected into the scar to replace the damaged tissue or to induce repair by resident stem or progenitor cells. An intraco- ronary, transendocardial, transpericardial, or intramyocardial route of administration may be chosen. An intravenous route of administration is not preferred because of cells homing to non-targeted organs. MIAMI cells may differentiate into endothelial cells, then into cardiomyocytes; alternatively, they can induce differentiation of resident stem or progenitor cells by paracrine effects. Vascular maturation (e.g., arteriogenesis and collateral growth) may be promoted by further administration of MIAMI cells or differ- entiated cells to the subject, or in situ differentiation and proliferation of resident circulating cells of the subject. An increase in ejection fraction, a decrease in infarct size, a reduction in number or severity of symptoms, replacement of scar tissue, angiogenesis, neovascularization, or a combination thereof would indicate effective treatment.

In other embodiments, MIAMI cells or differentiated cells are administered locally on a support medium. One specific, non-limiting example of a support medium is a sterile mesh, or matrix, upon which the MIAMI cells are cultured. In one embodiment, the support medium is a biodegradable mesh. In another embodiment, the support medium is not biodegradable. The size of the mesh, and the density of cells on it, can vary depending on the defect being treated. In another embodiment, the cells are encapsulated prior to administration, such as by co-incubation with a biocompatible matrix or gel known in the art. A variety of encapsulation technologies have been developed (for example, Lacy et al., Science,

254: 1782-1784, 1991 ; Sullivan et al, Science, 252:718-721 , 1991 ; WO 91/10425; WO 91/10470; U.S. Patent 4,892,538; U.S. Patent 5,01 1 ,472; U.S. Patent 5,837,234).

The cells can be repeatedly administered at intervals until a desired therapeutic effect is achieved. In certain embodiments, MIAMI cells may be isolated from the subject requiring treatment to avoid rejection of administered cells. But allogeneic transplantation is preferred. Examples of treatment with other types of mesenchymal stem cells include Amado et al {Proc. Natl. Acad. Sci. USA, 102: 11474-1 1479, 2005) and Berry et al. (Am. J. Physiol, 290:H2196-H2203, 2006). They are helpful because they illustrate how MIAMI cells can be used in clinical trials. Isolated MIAMI cell can be transduced using standard procedures known in molecular biology in order to introduce a nucleic acid molecule of interest into the cell (e.g., agents like lipids or liposomes, biolistics, viral membrane lipids and proteins). The nucleic acid molecule may encode a polypeptide. The polypeptide encoded by the nucleic acid molecule can be from the same species as the cells (homologous), or can be from a different species (heterologous). For example, a nucleic acid molecule can be utilized that supplements or replaces deficient production of a peptide by the tissue of the host wherein such deficiency is a cause of the symptoms of a particular disorder. In this case, the cells act as a source of the peptide. In other embodiments, the nucleic acid molecule may be an antisense molecule, a ribozyme molecule, or an siRNA molecule. In some embodiments, the nucleic acid sequence of interest is operably linked to one or more regulatory elements, such as a transcriptional and/or translational regulatory element. Regulatory elements include elements such as a promoter, an initiation codon, a stop codon, mRNA stability regulatory elements, and a polyadenylation signal. A promoter can be a constitutive promoter or an inducible promoter. Specific non-limiting examples of promoters include those from genes encoding endothelin-1 , ICAM-2, PECAM-I , P-selectin, Tie-l/Tie-2, von Willebrand factor, VEGF receptor 2, VE-cadherin, and contractile proteins specific for cardiac muscle, and promoters including the TET-responsive element for inducible expression of transgene. In another embodiment, the nucleic acid sequence of interest is inserted into a vector, such as an expression vector. Procedures for preparing expression vectors are known to those of skill in the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., CSHL Press, 1989. Expression of the nucleic acid of interest occurs

when the expression vector is introduced into an appropriate host cell. The vector can be a viral vector, such as an adenoviral or retroviral vector.

Retroviral vectors of use are produced recombinantly by procedures already taught in the art. For example, WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines. The techniques used to construct vectors, and transfect and infect cells are widely practiced in the art. Examples of retroviral vectors are those derived from murine, avian or primate retroviruses. Retroviral vectors based on the Moloney (Mo) murine leukemia virus (MuLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells. Other suitable vectors include those based on the Gibbon Ape Leukemia Virus (GALV) or lentiviruses like HIV.

In producing retroviral vector constructs derived from the Moloney murine leukemia virus (MoMLV), in most cases, the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are usually expressed under the control of the strong viral promoter in the LTR. Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences. Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced. Alternatively, the packaging cell line harbors an integrated provirus. The provirus has been crippled so that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. Instead, RNA produced from the recombinant virus is packaged. The virus stock released from the packaging cells thus contains only recom- binant virus.

The range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein. The recombinant virus can be used to infect

virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product. In general, murine ecotropic env of MoMLV allows infection of rodent cells, whereas amphotropic env allows infection of rodent, avian and some primate cells, including human cells. Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PAl 2 and PA317 (Miller et al, MoI. Cell. Biol., 5:431- 437, 1985; Miller et al., MoI. Cell. Biol, 6:2895-2902, 1986; Danos et al, Proc. Natl Acad. ScL USA, 85:6460-6464, 1988). Xenotropic vector systems exist which also allow infection of human cells (U.S. Patent 5,638,928).

According to this example, cells are cultured in vitro as described herein and an exogenous nucleic acid is introduced into the cells by any method known to one of skill in the art, for example, by transfection or electroporation. The transfected cultured cells can then be studied in vitro or can be administered to a subject. Methods for the intro- duction of nucleic acid sequences into multilineage-inducible cells are known in the art (e.g., see U.S. Patent 6,110,743).

VII. Use of MIAMI Cells to Screen for Agents that Induce Differentiation

Methods are provided for screening agents that induce differentiation of multi- lineage inducible cells. According to this method, a population of MIAMI cells is produced as described above. The population of cells is contacted with an agent of interest, and the effect of the agent on the cell population is then assayed. Differentiation of some or all of the MIAMI cells identifies the agent as a differentiation-inducing agent. An agent may induce MIAMI cells to differentiate into cells of endothelial lineage or to undergo cardiomyogenic differentiation. Differentiation of MIAMI cells contacted with an agent can be assessed by any means known to one of skill in the art, including those methods described herein. Such methods include, without limitation, morphological, biochemical, or functional determinations.

In one embodiment, the morphology of MIAMI cells contacted with a test agent is examined, for example by light, electron, or epifluorescent microscopy. In some embodiments, physiologic assays are performed, for example, resting membrane potential, depolarization-induced currents, or electrical coupling by cardiac gap junctions

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may be measured in cells undergoing cardiomyogenic differentiation in response to an agent. In other embodiments, the products of transcription of marker genes into RNA or their RNA translated into protein are used to assess differentiation. Suitable assay methods include, but are not limited to in situ hybridization, Northern analysis, ribo- nuclease protection, RT-PCR, ELISA, epifluorescence, and Western blot.

In one embodiment, MIAMI cells contacted with the agent are compared with a control. Suitable controls include MIAMI cells that are not contacted with the agent, or contacted with vehicle alone. Standard values can also be used as a control.

VIII. Kits

The cells described herein are ideally suited for the preparation of a kit. The kit can include a carrier means, such as a box, a bag, or plastic carton. In one embodiment, the carrier contains one or more containers such as vials, tubes, and the like that include a sample of MIAMI cells. In another embodiment, the carrier includes a container with an agent that affects differentiation, a buffer, or a vehicle for the introduction of the cells. Instructions can be provided to detail the use of the components of the kit, such as written instructions, video presentations, or instructions in a format that can be opened on a computer (for example, a diskette or CD-ROM disk). These instructions indicate, for example, how to administer the cells to treat a disease or other disorder.

The following examples are provided to illustrate certain particular features and/ or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

EXAMPLES

ISOLATION AND CHARACTERIZATION OF MIAMI CELLS

The isolation, characterization, and expansion of multilineage-inducible cells obtained from post-natal, human bone marrow are demonstrated under varying low- oxygen conditions.

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1. Introduction

Although it is becoming more evident that primitive pluripotent stem cells are present in the bone marrow (BM), very little is known regarding the mechanisms that regulate sternness (the capacity of the most primitive stem cells to preserve their full pluripotential without differentiating), self-renewal, and maturation.

All nucleated cells in the human body sense O ₂ concentration and respond to reduced O ₂ availability (hypoxia). The ability of cells to respond to hypoxia is essential for both normal development and postnatal physiology. Adaptive responses to physiologic stimuli involve changes in gene expression. Hypoxia-inducible factor- 1 (HIF-I), a transcriptional factor that activates more than 40 genes, has been shown to play an essential role in a variety of cellular and systemic homeostatic responses to hypoxia.

The process of growing cells in culture was first developed almost one hundred years ago. Since then, tissue culture for in vitro studies has become a complex science, although at the present time in vitro cell cultures are usually performed in incubators in the presence of 5% CO ₂ (to maintain appropriate pH conditions) and ambient air. Ambient air is composed of 78% N ₂ , 21% O _2, and small quantities of CO ₂ , argon, and helium. However, the physiologic oxygen pressure inside the human body is much lower and varies from tissue to tissue, ranging from 1 % in cartilage and bone marrow to a maximum of 10-13% in the arteries, lungs, and liver. Marrow stem cells are routinely isolated from BM and cultured in tissue culture incubators, where the partial pressure of the atmospheric oxygen (pθ ₂ ) at sea level is 159 mm Hg, corresponding to -21% O ₂ . In contrast, the pθ ₂ in the BM is much lower, between 1% and 7%.

The first studies of low pθ ₂ in tissue cultures demonstrated a positive effect on cell growth. These results were followed a few years later by the work of Andrew et al., who showed that connective tissue cultured in the presence of high pθ ₂ promoted bone formation, whereas low oxygen tension favored cartilage development. More recently, the importance of oxygen tension has been recognized in cancer research, where the microenvironment of solid tumors has been shown to contain regions poor in oxygen. Other studies point to a fundamental role for oxygen tension in metastatic progression. The role of low pθ ₂ in stem cells has been most widely studied with hematopoietic stem cells (HSC). Oxygen has a physiological function in the BM microenvironment, particularly as a regulator of the balance between sternness and differentiation. It is

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known that in BM the hematopoietic progenitor cells are not randomly distributed throughout the marrow space. Committed precursors are located close to the blood vessels - i.e., in better-oxygenated areas - while more immature progenitors reside preferentially in areas of lower oxygen tension. Scho field {Blood Cells, 4:7-25, 1978) proposed a model for the HSC population, suggesting that they would likely proliferate without limit when they were located in a specific area he called a stem cell niche. A cell in such an environment would not differentiate, while daughter cells that moved outside the influence of the niche would differentiate. Moreover, Cipolleschi et al. proposed that the "stem cell niche" is an area of extreme low oxygen tension, in which only oxygen-independent cells are able to survive.

Based on these studies we examined the effect of low oxygen tension on the growth of BM-derived MIAMI cells. Very little is known about the role of pθ ₂ in marrow stroma-derived cells, compared to what is known about pθ ₂ in hematopoietic stem cells. Recently, it was reported that cultures of rat MSC at low pθ ₂ (5% O ₂ ) had a greater number of cells and generated more bone when implanted into rats compared with cultures grown at 21% O ₂ .

Here, we report studies on the effect of low pθ ₂ on cell culture of MIAMI cells with respect to levels of cell viability and proliferation, as well as their potential for self-renewal (maintenance of expression of human embryonic stem cell markers) and osteogenic differentiation (development of expression of osteoblastic cell markers). MIAMI cells grown at low pθ ₂ increased the expression of embryonic stem cells markers Oct-4, Rex-1, SSEA4, and telomerase reverse transcriptase (hTERT), even under in vitro conditions that direct osteoblastic differentiation. Cell proliferation was augmented, and differentiation toward the osteoblastic lineage was inhibited. The details of these studies are described below.

2. Materials and Methods

Human bone marrow isolation and procedures

Bone marrow (BM) cells were isolated from human vertebrae of six donors, four males and two females (3 to 55 years old), immediately after death from traumatic injuries as previously described, according to guidelines of the University of Miami

Committee on the Use of Human Subjects in Research. Hepatitis B and C viruses and

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human immunodeficiency virus type 1 were ruled out by history and lab tests on BM specimens. Vertebral bodies were removed from normal, apparently healthy donors within two hours after their hearts stopped beating. Unfractionated bone marrow, including adherent and nonadherent cells, was plated (without prior gradient centrifuga- tion, immunoselection, or immunodepletion) at a constant density of 10 ⁵ cells/cm ² in fibronectin-coated 15-cm dishes in low-glucose DMEM (Invitrogen, Grand Island, NY) with 5% fetal bovine serum (FBS, HyClone Laboratories, Logan, UT) as previously described. Bone marrow nucleated cells were counted with a hemacytometer using 4% acetic acid and 0.4% trypan blue (Sigma, St. Louis, MO).

Culture of MIAMI cells

The isolation of MIAMI cells was described in WO 2004/069172 and US 2006/ 0147426. Briefly, unfractionated BM cells were plated at 1 x 10 ⁵ /cm ² in T75 flasks (Costar, Cambridge, MA) in the presence of low-glucose DMEM, 5% FBS, 100 U/ml penicillin (Invitrogen), and 1 mg/ml streptomycin (Invitrogen). The cells were incubated in a 100% humidified atmosphere of 3% O ₂ , 5% CO ₂ , and 92% N ₂ . Half of the culture medium was changed after a week; thereafter, half the medium was replaced twice a week. MIAMI cells were cultured up to 30% confluence. For expansion, MIAMI cells were replated at a density of 2,000 cells/cm ² in 98% low-glucose DMEM, 2% FBS, 5 ng/ml human EGF, 5 ng/ml human PDGF-BB, and 100 U penicillin/ 1000 U streptomycin (expansion medium) at various oxygen tensions (pθ ₂ , see below), with medium changed twice a week. In our studies, we utilized MIAMI cells from passages 1 to 6.

Culture of marrow stromal cells

Marrow stromal cells from a 23-year-old male healthy donor were purchased from Cambrex (Walkersville, MD). Cells were plated at 6 x 10 ⁴ /cm ² in T75 flasks (Costar) in the presence of low-glucose DMEM, 10% FBS, 100 U/ml penicillin (Invitrogen), 1 mg/ml streptomycin (Invitrogen). The cells were incubated in a 100% humi- dified atmosphere of 21% O ₂ and 5% CO ₂ . Medium was changed twice a week.

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Oxygen tension incubation

To test the effects of oxygen tension on our cells, various pθ ₂ were employed: 1%, 3%, 5%, 10%, or 21% O ₂ . Cells in culture plates/flasks were placed into hermetic chambers (Billups-Rothenberg, Del Mar, CA) into which the appropriate gas mixture was infused for 3 min twice a day. Oxygen tension (1%, 3%, 5%, 10%, or 21% O ₂ ) was controlled by regulating the N ₂ concentration. The hermetic chambers were placed into a regular incubator at 37°C. Once the optimal oxygen concentration (3%) was empirically determined, all subsequent experiments were performed at 3% O ₂ , with 5% CO ₂ and 92% N ₂ in a tri-gas incubator (ThermoForma, Waltham, MA).

Cell growth assay

Proliferation of MIAMI cells was assessed at various pθ ₂ to identify optimal conditions. The cells were plated onto 60-mm dishes, in triplicate in the presence of expansion medium, at 1 ,500 cells/cm ² . MIAMI cells were incubated at various pθ ₂ as indicated above for the. At the end of assay days 3, 7, 10, and 15, cells were rinsed with PBS, detached with trypsin-EDTA, and then counted with a hemacytometer.

[* H] Thymidine incorporation

Thymidine incorporation was assessed by plating 2 x 10 ⁴ cells/well onto 24- well plates in the presence of expansion medium. Plates were incubated at either 21% or 3% O ₂ for 24, 48, or 72 hours. At the end of each assay time cells were rinsed twice with PBS, serum-free medium, and 1 μCi [ ³ H]-thymidine (a specific activity of 30.0 Ci/mmol, Amersham Biosciences, Piscataway, NJ) were added, and then the cells were incubated for an additional 24 hours at either 21% or 3% O ₂ . At the end of the incuba- tion, the medium was removed, and the cells were rinsed twice with cold PBS and then incubated withl mL of cold (4°C) 5% trichloroacetic for 30 min. Thereafter, the cells were rinsed twice with cold PBS, and 1 mL of 0.5 N NaOH was added to each well. The cell lysates were transferred into scintillation tubes. Incorporation of radioactive thymidine into DNA was detected by scintillation counting.

RNA isolation and RT-PCR

Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to provide semi -quantitative data on the expression of embryonic markers Oct-4, Rex-1, hTERT, and HIF-I α (a transcription factor specific for regulation of the gene expression of a spectrum of proteins for the cellular response to hypoxia).

At the indicated times, the medium was removed and total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA was resuspended in DEPC-treated water, and its concentration was determined by spectrophotometry. For RT-PCR analysis, total RNA was treated with RQl RNase-free-DNase, and then RNA was precipitated in a 2 M LiCl solution. Five μg of high-molecular-weight RNA was reverse transcribed using MuLV reverse transcriptase, 200-pmol random hexamer primer, and 50 pmol of oligo(dT). Aliquots (4%) of the total cDNA were amplified in each PCR in a 20 μl reaction mixture containing 10 pmol of 5' and 3' primers in a standard PCR buffer. Amplifications were performed in a GeneAmp9600 thermal cycler for 23-30 cycles (typically: 94°C for 30 sec; 58 ⁰ C for 45 sec; 72 ⁰ C for 60 sec) after an initial denaturation of 94 ⁰ C for 2 min. Amplification products in a 10 μl aliquot were size separated by electrophoresis in agarose gel. Semiquantitative analysis of gene expression was assessed from the differences between the amplified products after specific numbers of amplification cycles, selected during the logarithmic amplification phase and normalized to the corresponding elongation factor-1 alpha (EF-I α) amplification product after 23 amplification cycles. Amplification reactions were normally performed in duplicate or triplicate, and each experiment was repeated two or more times.

Flow cytometry analysis

MIAMI cells were plated in triplicate on 60-mm dishes in the presence of expansion medium at 3% and 21% pθ ₂ . Fifteen days later the cells were trypsinized and 1 x 10 ⁶ cells were aliquoted into FACS tubes (BD Bioscience, Palo Alto, CA). Cells were rinsed twice with a cold buffer solution (DPBS, 1% FBS, at pH 7.4) and incubated with the primary antibodies, SSEA4 or appropriate isotype-matched controls (R&D Systems, Minneapolis, MN) for 30 min at 4°C. Subsequently, cells were rinsed three times with a cold buffer solution and then incubated with the secondary antibody

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goat anti-mouse FITC conjugated (BD Bioscience). Cells were rinsed again twice with the cold buffer solution and fixed with 1% paraformaldehyde until analysis with a FACScan (BD Bioscience).

TRAP assay

Telomeric repeat amplification protocol (TRAP) assay was performed with a TRAP assay kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. Briefly, cell pellets were resuspended with the kit lysis buffer, CHAPS. Fifty microliters of reaction mixture containing 2 μl of protein extract were incubated at room temperature for 30 min and then subjected to 33 PCR cycles (94°C for 30 sec, 59 ⁰ C for 30 sec, 71 ⁰ C for 1 min). Twenty-five microliters of TRAP product were analyzed by electrophoresis in 0.5x Tris-borate-EDTA buffer on 12% polyacrylamide nondenaturing gels and visualized with ethidium bromide (Sigma) to stain DNA.

Statistical analysis

Statistical analyses were performed using Student's Mest, with /? < 0.05 considered significant.

3. Results Low oxygen tension increases the proliferation of MIAMI cells

To determine the most suitable oxygen concentration for the proliferation of MIAMI cells, cells were grown at five different oxygen tensions: 1, 3, 5, 10, or 21% O ₂ (air). At all pθ ₂ levels lower than air, we observed a significant increase in cell number compared to air at all days tested: 7, 10, and 15 days (Fig. IA). Moreover, cells grown at 3% O ₂ consistently showed a significant increase compared to the other pθ ₂ , from day 3 to day 15 (Fig. IA). Incubating the cells at 3% O ₂ increased the number of cells by more than 2 fold on all assay days compared to air. In the remainder of the experiments described here we used an oxygen concentration of 3% (hereafter named low pθ ₂ ) obtained with the tri-gas incubator described above.

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Low oxygen tension increases DNA synthesis in MIAMI cells

We next investigated whether the rate of [ ³ H]-thymidine incorporation (as an index of DNA synthesis and proliferation) differed in cells grown in air compared to those grown in low pθ ₂ . MIAMI cells were grown in air and low pθ ₂ in expansion medium in the presence of [ ³ H] -thymidine for various periods of time. At all times analyzed cells grown in low pθ ₂ incorporated more [ ³ H]-thymidine than cells grown in air (Fig. IB). On day 1 the level of incorporation was two-fold higher in low pθ ₂ than in air; on day 2 the increase was almost three-fold higher than in air; and on day 3 the incorporation was almost five times more (Fig. IB). These data strongly suggest that at low pθ ₂ each cell-cycle phase (population doubling time) is of shorter duration compared to cells grown in air.

Low oxygen conditions enhance the maintenance of primitive stem cell state

To assess whether oxygen tension affects the ability of MIAMI cells to differen- tiate, cells were grown in the presence of expansion and osteogenic medium in air and low pθ ₂ . Semiquantitative RT-PCR analysis was utilized to evaluate markers characteristic of primitive embryonic stem cells. MIAMI cells grown at low pθ ₂ showed up- regulation of the expression of the embryonic transcription factors Oct-4 (one of the earliest genes known to be expressed during mammalian embryogenesis), Rex-1 (expressed in the inner cell mass of blastocyst), and hTERT (the specialized reverse transcriptase that synthesizes telomeric repeats) (left panel of Fig. 2A). Moreover, MIAMI cells grown at low pθ ₂ maintained the expression of these embryonic stem cell markers even in the presence of osteoblastic differentiation medium (right panel of Fig. 2A). In addition, flow cytometry analysis was utilized to study the expression of the surface marker stage-specific embryonic antigen 4 (SSEA4), highly expressed in early human embryonic stem cells. MIAMI cells maintained at low pθ ₂ showed 70% of SSEA4 expression compared to cells grown at 21% O ₂ where only 22% was noticed (Fig. 2B). Furthermore, we examined telomerase activity, a functional assessment of stem cell status, by determining the length of telomere repeats at low oxygen tension. TRAP assays showed the expected DNA ladder with six nucleotide increments, telomerase products, in MIAMI cells grown at low oxygen (3%) tension (lane 3 of Fig. 3) and are detected at levels comparable to those observed in telomerase-immortalized human

foreskin fibroblast (hTERT-BJl ; lane 2 of Fig. 3). These results suggest that low pθ ₂ strongly promotes the maintenance of MIAMI cells in a more undifferentiated stem- like state.

Low oxygen tension transiently upregulates hypoxia-inducible factor- Ia (HIF-Ia) expression

HIF- lα is a transcription factor that plays an essential role in O ₂ homeostasis and is activated by hypoxia. HIF- lα activates more than 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, heme oxygenase- 1 , inducible nitric oxide synthase, transferrin, and vascular endothelial growth factor. Semiquantitative RT-PCR analysis showed that HIF-I α is upregulated in MIAMI cells within two hours at low pθ ₂ .

4. Discussion We demonstrated for the first time that oxygen tension plays an essential role in regulating the balance between self-renewal and differentiation of human marrow stroma-derived cells. Although the effects of low oxygen concentration on cell growth have previously been examined by others in different cell models, we are reporting the first study on human marrow-derived stromal stem cells such as the MIAMI cells. Our results demonstrate that low oxygen concentration increases cell proliferation, with a maximal difference observed on MIAMI cells maintained at 3% O ₂ . Low pθ ₂ also inhibits osteoblastic differentiation, even if MIAMI cells are maintained under osteoblastic differentiation medium conditions for up to 4 weeks. Moreover and importantly, low pθ ₂ is proven to be a cell culture condition that is critical in order to main- tain MIAMI cells in a less-differentiated state. Specific key markers characteristic of embryonic stem cells were upregulated and maintained under expansion conditions set at 3% O ₂ . Therefore, oxygen concentration is likely to be a key condition regulating the balance between self-renewal and differentiation of other stem or progenitor cells.

Compared to air, all low pθ ₂ examined (1 , 3, 5, or 10%) consistently showed an increased number of cells for at least a period of 15 days (Fig. 1). Although 3% O ₂ had the most dramatic effect, all the other lower-than-air pθ ₂ concentrations also showed increased proliferation rates. Cells exposed to pθ ₂ as low as 1% consistently showed

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better proliferation rates than 21% during this time period. Thus, it is likely that the pθ ₂ may influence the behavior of the cell in different ways and that lineage-specific differentiation of stem cells may be enhanced when exposed to a specific pθ ₂ range. Similarly, a pθ ₂ lower than 3% may further favor stem cell self-renewal and maintenance over increased proliferation, since specific anatomical sites in the BM are estimated to have pθ ₂ as low as 1%. As mentioned previously, ambient air containing 21% O ₂ , the standard condition for cell culture growth, is not a physiological condition for any kind of cell in the human body. Oxygen concentration in human tissues ranges from 1 % in the BM and cartilage to 13% in the liver, lungs, and other organs. It has been well established that high oxygen concentrations could be toxic through the generation of reactive oxygen species (ROS), a process by which excess electrons are transferred to oxygen, leading to the noxious oxidation of adjacent molecules. Although we have not directly addressed the formation of ROS in MIAMI cells grown in air, in the human body ROS are formed at O ₂ concentrations much lower than 21%, suggesting this as a potential toxic effect.

To determine the mechanisms by which low oxygen influences cell self-renewal vs. differentiation, several experiments were performed. Cell-cycle analysis did not show any significant differences in the number of cells detected at any phase of the cell cycle, at either 3% or 21% oxygen culture conditions, suggesting that oxygen tension has no effect on the progression of MIAMI cells through the cell cycle. However, [ ³ H]- thymidine incorporation (as an index of DNA synthesis) demonstrated a significant increase in cell proliferation at low pθ ₂ compared to air (Fig. IB). On the other hand, preliminary studies suggested no detectable effect of low pθ ₂ on apoptosis. Thus, it appears that compared to air, cells are able to progress faster through a normal cell cycle at low pθ ₂ , resulting in the population doubling time being reduced at low pθ ₂ . This finding could explain, at least in part, the higher cell numbers seen at low pθ ₂ . Further, the increased proliferation takes place with concomitant increased expression of markers characteristic of embryonic stem cells (Fig. 2) as well as sustained telome- rase activity as demonstrated by the length of telomere repeats (Fig. 3). These results show that low pθ ₂ not only increases proliferation, but also maintains the dividing cells in a relatively undifferentiated state, demonstrating that low pθ ₂ is a key condition for promoting stem cell self-renewal.

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A widely accepted mechanism of oxygen tension-mediated regulation of a cell's physiology is the activation of the transcriptional factor HIF- lα, which in turn regulates the transcription of a number of downstream target genes. We observed that HIF- lα was upregulated in MIAMI cells exposed to low pθ ₂ (3%) within 120 min, while the housekeeping gene ELF- lα was unaffected. Furthermore, key markers prototypic of human embryonic stem cells were also upregulated under similar conditions (Fig. 2). These results suggest, though do not prove, a potential link between HIF- lα upregula- tion and increased Oct-4, Rex-1, hTERT, and SSEA4 expression in response to decreased pθ _2. In turn, such a relationship could represent a possible mechanism of pθ ₂ regulation of cellular physiology restricted to undifferentiated stem cells.

Consistent with our data, Lennon et al. (J. Cell Physiol, 187:345-355, 2001) reported that proliferation of rat marrow stromal cells increased at low pθ ₂ (5%) compared to air. In contrast with our data, howver, these researchers reported that extracellular mineralization also increased in cell cultures maintained at low pθ ₂ compared to air. The apparent discrepancy in results may be due to differences between human and rat cells and that MIAMI cells may represent a more undifferentiated population of marrow stromal cells, or both.

Our data are consistent with the notion of the "niche" model elaborated by Schofield, where stem cells or more developmentally primitive cells in the BM are located within a specific area characterized by a unique microenvironment, low in pθ ₂ , that contributes to maintain a limitless potential of stem cells to self-renew (proliferate without differentiation). The positive effect of low pθ ₂ on maintaining sternness has also been shown in other types of stem cells. Epithelial stem cells from placenta, the cytotrophoblasts, proliferate under low oxygen tension (2%), while at 21% oxygen they differentiate to form syncytiotrophoblasts. Furthermore, the growing embryo is little vascularized during the first weeks of development, with an oxygen tension of the inter- villus space of about 2.3%. The low oxygenation allows the immature embryo cells to grow. Moreover, in vitro fertilization techniques are improved when low oxygen tension is used to promote embryo development. A recent in vitro study on embryonic stem cells (ES) reported that ES better maintain full pluripotency at low pθ ₂ .

Another mechanism by which low pθ ₂ may regulate self-renewal vs. differentiation involves the induced production of specific growth factors and cytokines and

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their respective receptors. Factors such as platelet-derived growth factor-B (PDGF-B) and PDGF receptor, and vascular endothelial growth factor (VEGF) have all been reported to increase in response to low pθ ₂ in specific cells. Furthermore the hepato- cyte growth factor (HGF) receptor, c-Met, is also upregulated by low pθ ₂ . In conclusion, oxygen tension can regulate the balance between self-renewal and differentiation of multilineage-inducible cells: 3% pθ ₂ is a normoxic, instead of a hypoxic, condition for the MIAMI cells to maintain their sternness after in vitro expansion. The use of low pθ ₂ levels to enhance the in vitro survival and/or self-renewal of human MIAMI cells, and perhaps other stem cells, represents an important advance in stem cell research that could be critical for obtaining the great number of cells that would be needed for therapeutic application in the clinical arena.

DIFFERENTIATION OF ENDOTHELIAL CELLS FROM MIAMI CELLS

The differentiation and characterization of endothelial cells obtained from the MIAMI cells were demonstrated under in vitro culture conditions.

Endothelial-Induction Conditions

To culture MIAMI cells, they were replated at a density of 2.5 x 10 ⁴ cells/cm ² in fibronectin-coated culture vessels, in low-glucose DMEM supplemented with 3% FBS, 100 μM ascorbic acid 2-phosphate, and Ix lipid solution (188 nM linoleic acid, 400 nM lipoic acid, 12.9 nM arachidonic acid, 1.12 μM cholesterol, 290 nM DL-α tocopherol- acetate, 69.9 nM linolenic acid, 85.9 nM myristic acid, 69.4 nM oleic acid, 76.5 nM palmitic acid, 77.1 nM palmitoleic acid, and 68.9 nM stearic acid).

MIAMI cells were cultured under four differentiation conditions: control (C), only VEGF (V), growth factor (GF), or EGM-2 (EGM) from Cambrex/Clonetics (Walkersville, MD). The control is expansion medium containing low-glucose DMEM, 3% FBS, 100 μM ascorbic acid 2-phosphate, Ix insuling-transferrin-selenium (Sigma), and Ix lipid solution (188 nM linoleic acid, 400 nM lipoic acid, 12.9 nM arachidonic acid, 1.12 μM cholesterol, 290 nM DL-α tocopherol-acetate, 69.9 nM linolenic acid, 85.9 nM myristic acid, 69.4 nM oleic acid, 76.5 nM palmitic acid, 77.1 nM palmitoleic acid, and 68.9 nM stearic acid), in which only half the medium is replaced with fresh medium every 3-4 days. VEGF medium contains low-glucose DMEM, 100 μM

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ascorbic acid 2-phosphate, 100 U penicillin, 1 ,000 U streptomycin, Ix lipid solution, Ix insuling-transferrin-selenium, and 50 ng/ml VEGF (Sigma). Growth factor medium contains low-glucose DMEM, 100 μM ascorbic acid 2-phosphate, Ix lipid solution, Ix insuling-transferrin-selenium, 50 ng/ml VEGF, 10 ng/ml bFGF (Sigma), 10 ng/ml EGF (Sigma), 10 ng/ml IGF-I (Sigma), and 100 nM hydrocortisone. EGM-2 medium is a proprietary formulation containing ascorbic acid, GA- 1000 (gentamicin, amphotericin- B), 5% FBS, vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor- 1 , and hydrocortisone at unknown concentrations.

Human microvascular endothelial cells (HMVEC) from Cambrex/Clonetics were plated at 1 x 10 ⁴ cells/cm ² in culture vessels in EGM-2 medium. These cells are used as positive controls.

Cultures were maintained by media exchange at 3-day to 4-day intervals. Cells were treated for 5 days, 10 days, or 21 days before assessment of endothelial marker expression.

TABLE 2. Primer Pairs (SEQ ID NOS: 1-6, respectively) used in qRT-PCR to assess expression of endothelial markers

Fig. 4 shows the expression of endothelial markers during culture of MIAMI cells in an endothelial-induction medium. Expression of CD31 (Fig. 4A), CD36 (Fig. 4B), and von Willebrand factor (vWF) (Fig. 4C) was substantially increased only after 21 days of culture in differentiation medium containing VEGF, bFGF, EGF, IGF-I , and hydrocortisone.

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DIFFERENTIATION OF CARDIOMYOCYTES FROM MIAMI CELLS

The differentiation and characterization of cardiomyocytes obtained from the MIAMI cells are demonstrated under in vitro culture conditions.

Cardiomyogenic Conditions

MIAMI cells from the fourth to fifth passage were replated at a density of 1 x 10 ⁴ cells/cm ² in expansion medium containing 2% FCS, lO ng/ml EGF, lO ng/ml PDGF-BB, 3 μM 5-azacytidine, 1 nmol/L insulin, and 10 ng/ml TGFβl for 24 hours. The medium was changed at two-day intervals for a week, and the cells were incubated with 5-azacytidine for another 24 hours. At different times during the induction protocol, cells were evaluated for expression of cardiomyocyte markers by qRT-PCR.

TABLE 3. Primer Pairs (SEQ ID NOS:7-14, respectively) used in qRT-PCR to assess expression of cardiomyocyte markers

Fig. 5 shows the expression of endothelial markers during culture of MIAMI cells in a cardiomyogenic medium. There was some expression of MYL-2 (Fig. 5A), MYL-7 (Fig. 5B), TNNT2 (cTnT, Fig. 5C), and TNNI3 (cTnl, Fig. 5D) sometime after

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3 days of culture in differentiation medium containing FCS, EGF, PDGF-BB, 5-aza- cytosine, insulin, and TGFβl .

MIAMI cells clearly express markers found among stem-like cells. Moreover, MIAMI cells are equipped to respond to signals leading to differentiation toward diverse lineages. Like hMSCs and MAP cells, MIAMI cells can be differentiated to cells that express markers unique to endothelial cells and cardiomyocytes. Although mesodermal-derived lineages could be obtained using primary marrow stromal stem cells isolated by standard procedures for isolating mesenchymal stem cells (e.g., Pittenger et al, Science, 284:143-147, 1999), neural and endodermal differentiation could be obtained only with MIAMI cells. Use of differentiated cells would reduce the potential risk of unbridled proliferation of MIAMI cells in patients during initial safety studies and channel their potential into relevant lineages.

When MIAMI cells were cultured at high density and exposed to agents that induce their differentiation, proliferation slowed to a stop and terminal differentiation was evident on both a molecular and functional basis. Long-term culture (>30 days) of differentiated cells led in many cases to apoptosis with no evidence of transformation. But no Hayflick limit was seen if cells were kept at low density and under low oxygen conditions. We have now extended these results to differentiation of MIAMI cells into endothelial-like cells and cardiomyocyte-like cells.

USE OF MIAMI CELLS TO INDUCE NEOVASCULARIZATION

Peripheral vascular disease is a major health care problem in an aging society and its incidence in the US is over 300,000 patients per year. The natural capacity of collaterals to remodel and enlarge to compensate for the reduced flow that occurs after occlusion of a major artery is rarely sufficient to restore maximal flow capacity to levels required under various stress-conditions. In such patients during the late stages of peripheral vascular disease, progression of tissue hypoperfusion results in ischemic ulceration and gangrene. Unfortunately, amputation is required in more than a third of these patients. Rapid revascularization of injured, ischemic, and regenerating organs is essential for the restoration of their physiological function. Several protein- and gene- based strategies have succeeded in enhancing collateral development in animal models of ischemia. Given that the natural response to tissue ischemia is such a complex

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process, the delivery of a single growth factor may be too simple an approach. Thus, a great deal of interest has arisen in the potential of cell-based strategies in augmenting collateral responses. In this study, we tested the ability of MIAMI cells as adult stem cells to induce vasculogenesis and improve ischemic limb function.

Hind-Limb Ischemia Model

The neovascularization capacity of MIAMI cells was investigated in a murine model of hind-limb ischemia in eight-week old C57BL/6 mice. Immunosuppression was performed by injecting cyclosporine at 20 mg/kg weight for two days before, and daily after inducing ischemia, for the entire period of the study.

To produce hind limb ischemia, animals were anesthetized and the proximal portion of the femoral artery including the superficial and the deep branches were ligated twice with 7-0 silk suture and all the branches (superficial and deep) distal to the ligation till the bifurcation into saphenous and popliteal arteries were resected according to institutionally approved protocols. After 24 hours, MIAMI cells (1 x 10 ⁶ ) suspended in PBS (60 μL) were injected intramuscularly at four different sites of the ischemic leg.

Vascularization evaluation Probable improvement of ischemic limb perfusion was evaluated by using laser doppler perfusion imaging (LDPI) and histological evaluation(counting capillary density) at one- week or two-week time points after the injection of undifferentiated MIAMI cells. Tracing of the injected cells were determined by immunofluorescence technique using anti-human mitochondrial specific antibody. Later tissue samples were examined for the expression of CD31 (PECAM) by double immunofluorescence technique to evaluate the in vivo transdifferentiation ability of cells to endothelial cells.

Results

MIAMI cells were detected in adductor muscle one week after injection by double immunoflorescent technique using anti- human-mitochondrial antibody labeled with a green dye. The same section was stained with anti-CD31 (PECAM) antibody labeled with a red dye to assess endothelial cell marker expression. The images were

merged to assess differentiation of MIAMI cells to endothelial cells in vivo. Some MIAMI cells were found not to express CD31. This suggests that they did not differentiate or differentiated to a different vascular lineage such as smooth muscle cells. Some MIAMI cells found to be CD31 -positive were detected within the context of blood vessels. It is not clear if CD31 -positive MIAMI cells are associated with new blood vessels or with pre-existing vessels undergoing repair or remodeling. Some of the CD31 -positive cells were found to be of host origin. The ratio of vascularization rat in the ischemic limb divided by vascularization in the normal limb was 0.49 ± 0.02 and 0.7 ± 0.13 at 7-days and 14-days post-injection, respectively, in the group (n = 5) treated with MIAMI cells. By contrast, in the culture medium only-treated group (i.e., negative control, n = 5) the ratio was markedly lower: 0.31 ± 0.05 and 0.36 ± 0.02 at 7- days and 14-days post-injection, respectively.

Conclusions We conclude that MIAMI cells can acquire features of endothelial cells in response to angiogenic factors in vitro. MIAMI cells injected in a murine limb ischemia model significantly improved blood flow as determined by LDPI. MIAMI cells may contribute to neovascularization via vascular repair, remodeling, or both.

USE OF MIAMI CELLS TO PREVENT CELL DEATH AND APOPTOSIS

These characteristics of MIAMI cells make them appropriate adult stem cells with which to develop therapies to prevent cerebral ischemic damage. To induce ischemia, a model of global cerebral ischemia after acute myocardial infarction was used: brain slices were exposed to oxygen-glucose deprivation (OGD) for 40 min. Slices were injected with MIAMI cells or culture medium (negative control) after one hour of ischemia. Each injection consisted of 2-3 μL medium containing about 7000 cells. Quantification of cell death in the hippocampal CAl region was conducted by using propidium iodide (PI) fluorescence staining. Injections of MIAMI cells significantly protected the CAl region of the hippocampus as compared to the culture medium-injected group (p< 0.001 ; one way ANOVA followed by Tukey's post-hoc test). The PI fluorescence values for the ischemia, medium-injected, and MIAMI cell injected groups were 59 ± 4 (n = 4), 76 ± 9 (n = 7), 39 ± 3 (n = 7), respectively, as

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shown in Fig. 6. Our results clearly demonstrate the efficacy of MIAMI cells in neuroprotection. This suggests that employing MIAMI cells might be of therapeutic value against cerebral ischemia.

In stating a numerical range, it should be understood that all values within the range are also described (e.g., one to ten also includes every integer value between one and ten as well as all intermediate ranges such as two to ten, one to five, and three to eight). The term "about" may refer to the statistical uncertainty associated with a measurement or the variability in a numerical quantity which a person skilled in the art would understand does not affect operation of the invention or its patentability.

All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. A claim which recites "comprising" allows the inclusion of other elements to be within the scope of the claim; the invention is also described by such claims reciting the transi- tional phrases "consisting essentially of (i.e., allowing the inclusion of other elements to be within the scope of the claim if they do not materially affect operation of the invention) or "consisting of (i.e., allowing only the elements listed in the claim other than impurities or inconsequential activities which are ordinarily associated with the invention) instead of the "comprising" term. Any of these three transitions can be used to claim the invention.

It should be understood that an element described in this specification should not be construed as a limitation of the claimed invention unless it is explicitly recited in the claims. Thus, the granted claims are the basis for determining the scope of legal protection instead of a limitation from the specification which is read into the claims. In contrast, the prior art is explicitly excluded from the invention to the extent of specific embodiments that would anticipate the claimed invention or destroy novelty.

Moreover, no particular relationship between or among limitations of a claim is intended unless such relationship is explicitly recited in the claim (e.g., the arrangement of components in a product claim or order of steps in a method claim is not a limitation of the claim unless explicitly stated to be so). All possible combinations and permutations of individual elements disclosed herein are considered to be aspects of the inven-

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tion. Similarly, generalizations of the invention's description are considered to be part of the invention.

From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.