Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof

This application claims the benefit of U.S. Provisional Application No. 60/149,849 filed on Aug. 19, 1999.

This invention relates to methods and compositions for the differentiation of stromal cells from adipose tissue into hematopoietic supporting stromal cells and myocytes of both the skeletal and smooth muscle types.

The neonatal period in human development is characterized by the presence of “stem” cells with the potential to develop along multiple differentiation pathways. The terminal differentiation of these cells is determined by cytokine and hormonal cues which co-ordinate organogenesis and tissue architecture. Murine embryonic stem cells have been isolated and studied extensively in vitro and in vivo. Using exogenous stimuli in vitro, investigators have induced ES cell differentiation along multiple lineage pathways. These include neuronal, B lineage lymphoid, and adipocytes (Dani et al. (1997) J. Cell Sci. 110: 1279; Remoncourt et al. (1998) Mech. Dev. 79:185; O\'Shea K S (1999) Anat. Rec. 257:32). The ES cells have been manipulated in vivo by homologous recombination techniques to generate gene specific null or “knock-out” mice (Johnson R S (1989) Science 245:1234). Once ES cell clones lacking a specific gene are isolated, they are transplanted into a fertilized murine zygote. The progeny of this isolated ES cell can develop into any and all murine tissues in a coordinated manner.

A stem cell must meet the following criteria: (1) ability of a clonal stem cell population to self-renew; (2) ability of a clonal stem cell population to generate a new, terminally differentiated cell type in vitro; and (3) ability of a clonal stem cell population to replace an absent terminally differentiated cell population when transplanted into an animal depleted of its own natural cells.

Multipotential stem cells exist in tissues of the adult organism. The best characterized example of a “stem cell” is the hematopoietic progenitor isolated from the bone marrow and peripheral blood. Seminal studies by Trentin, Till and McCulloch (McCulloch et al. (1996) Proc. Can. Cancer Conf. 6:356-366; Curry et al. (1967) J. Exp. Med. 125:703-720) examined lethally irradiated mice. In the absence of treatment, these animals died because they failed to replenish their circulating blood cells; however, transplantation of bone marrow cells from a syngeneic donor animal would rescue the host animal. The donor cells were responsible for reconstituting all of the circulating blood cells. A wealth of elegant studies have gone on to demonstrate that donation of a finite number of undifferentiated hematopoietic stem cells is capable of regenerating each of the eight or more different blood cell lineages in a host. This work has provided the basis for bone marrow transplantation, a widely accepted therapeutic modality for the treatment of cancer and inborn errors of metabolism in man. Thus, hematopoietic stem cells remain present in the normal human bone marrow throughout life; they are not limited to the neonatal period.

The recent development of entire organisms from a single donor cell are consistent with this hypothesis. The “Dolly” experiment showed that cells isolated from an ovine mammary gland could develop into a mature sheep (Pennisi & Williams (1997) Science 275:1415-1416). In similar murine studies, cells derived from the corpus luteum of the ovary could develop into a mature mouse (Pennisi (1998) Science 281:495). These studies suggest that stem cells with the ability to differentiate into any and all cell types continue to exist in the adult organism. Thus, “embryonic” stem cells may be retained throughout life.

In vitro experiments using cell lines of embryonic origin indicate that a mesodermal stem cell may exist. Work by Taylor and colleagues in the late 1970\'s demonstrated that murine embryonic fibroblasts such as C3H10T1/2 or 3T3 cells would differentiate along multiple mesodermal lineage pathways following exposure to 1 to 10 μM of 5′-azacytadine (Constantinides et al. (1977) Nature 267:364; Jones & Taylor (1980) Cell 20:85). Within 2 to 4 weeks, isolated clones displayed a morphology consistent with adipocyte, myocyte, chondrocyte or osteoblast differentiation. Biochemical data provided additional support for the identification of each of these lineages. This finding provided the basis for the identification of the master-regulatory transcription factor for skeletal muscle differentiation, myoD (Lassar (1986) Cell 47:649).

The adult bone marrow microenvironment is the potential source for these hypothetical mesodermal stem cells. Cells isolated from adult marrow are referred to by a variety of names, including stromal cells, stromal stem cells, mesenchymal stem cells (MSCs), mesenchymal fibroblasts, reticular-endothelial cells, and Westen-Bainton cells (Gimble et al. (1996) Bone 19:421-428). In vitro studies have determined that these cells can differentiate along multiple mesodermal or mesenchymal lineage pathways. These include, but are not limited to, adipocytes (fat cells) (Gimble et al. (1990) Eur. J. Immunol 20:379-386; Pittenger et al. (1999) Science 284:143-147; Nuttall et al. (1998) JBMR 13:371-382; Park et al. (1999) Bone 24:549-554), chondrocytes (cartilage forming cells) (Dennis et al. (1999) JBMR 14:700-709), hematopoietic supporting cells (Gimble et al. (1990) Eur. J. Immunol. 20:379-386), myocytes (skeletal muscle) (Phinney (1999) J. Cell. Biochem. 72:570-585), myocytes (smooth muscle) (Remy-Martin et al. (1999) Exp. Hematol. 27:1782-1795), and osteoblasts (bone forming cells) (Beresford (1989) Clin Orthop Res 240:270-280; Owen (1988) J. Cell. Sci. 10:63-76; Dorheim et al. (1993) J. Cell. Physiol. 154:317-328; Haynesworth et al. (1992) Bone 13:81-88, Kuznetsov et al. (1997) JBMR 12:1335-1347). The bone marrow has been proposed as a source of stromal stem cells for the regeneration of bone, cartilage, muscle, adipose tissue, and other mesenchymal derived organs. The major limitations to the use of these cells are the difficulty and risk attendant upon bone marrow biopsy procedures and the accompanying loss of memory B cells and hematopoietic stem cells with present harvesting procedures.

Another viable alternative to the use of bone marrow multipotential stem cells is adipose tissue. Adipose stromal cells provide an easily accessible and abundant source of stromal cells which can differentiate along multiple mesenchymal lineages. Methods and compositions are needed for the consistent and quantitative differentiation of adipose derived stromal cells into various cell types including for example hematopoietic stromal cells and skeletal and smooth muscle myocytes.

Compositions and methods for the differentiation of adipocytes are provided. Generally, the present invention provides methods and compositions for consistent and quantitative induction of stromal cells derived from subcutaneous, mammary, gonadal, or omental adipose tissues into the following fully differentiated and functional mesodermal cell lineages: hematopoietic supporting stromal cells, skeletal myocytes, and smooth muscle myocytes (myofibroblasts).

The compositions include a variety of chemical components which act as mitogens and differentiation inducing agents for the plated stromal cells and yield production of the desired cell type. The mitogens and inducing agents include, but are not limited to, interleukins, flt-3 ligand, stem cell factor, macrophage-colony stimulating factor, granulocyte-monocyte colony stimulating factor, erythropoietin, thrombopoietin, osteoprotegerin ligand, dexamethasone, hydrocortisone, 1,25 dihydroxy vitamin D₃, 2-mercaptoethanol, glutamine, 5′-azacytadine, amphotericin, transforming growth factor β and fibroblast growth factor.

The invention provides methods for determining the ability of these compositions to direct the differentiation and function of the adipose-derived stromal cells, for the transduction of viral vectors carrying regulatory genes into stromal cells, for the transfection of plasmid vectors carrying regulatory genes into stromal cells, for the tracking and detection of functional proteins encoded by these genes, and for developing biomechanical carriers for the re-introduction of these cells into living organisms.

The invention also provides methods and compositions which have utility in drug discovery for compounds and proteins with relevance to a wide spectrum of disease states including, but not limited to, aplastic anemia, muscular dystrophy, radiation poisoning, neuropathic muscular degeneration, urogenital malformations, and gastrointestinal malformations.

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