HK1238561B - Method of treating graft versus host disease - Google Patents

Description

Methods for treating graft-versus-host disease

This application is a divisional application of Chinese invention patent application No. 2011800422012, filed on July 4, 2011, and entitled “Method for treating graft-versus-host disease”.

field

The present invention relates to methods for enhancing the engraftment of hematopoietic progenitor cells, enhancing bone marrow transplantation, and preventing or reducing graft-versus-host disease. In one embodiment, the present invention relates to preventing or reducing graft-versus-host disease, a complication following allogeneic bone marrow transplantation in mammalian patients, particularly human patients.

background

A bone marrow transplant is required after a process that destroys the bone marrow. For example, after high-intensity whole-body radiation or chemotherapy, the bone marrow is the first target to fail. Metastatic cancers are often treated with very high-intensity chemotherapy that is intended to destroy the cancer but also effectively destroys the bone marrow. This necessitates a bone marrow transplant. However, except for the most acute, life-threatening conditions, utilizing a bone marrow transplant to alleviate any condition involving a bone marrow disorder is generally considered extremely risky due to the potential for the onset of graft-versus-host disease (GvHD).

GvHD is an immunological disorder that is a major factor limiting the success and availability of allogeneic bone marrow or stem cell transplants. GvHD is a systemic inflammatory response that causes a chronic disease and can lead to the death of the host animal. Currently, allogeneic transplants are invariably associated with a significant risk of GvHD, even when the donor and host are highly histocompatible.

GvHD is caused by donor T cells that react with incompatible host antigens distributed throughout the body, causing extremely strong inflammation. In GvHD, mature donor T cells that recognize the differences between the donor and the host are systemically activated. Current methods for preventing and treating GvHD involve administering drugs such as cyclosporine A and corticosteroids. They have serious side effects, must be administered for a long time, and are expensive to administer and monitor. Attempts have been made to use T cell removal to prevent GvHD, but this requires sophisticated and expensive facilities and expertise. Excessive levels of T cell removal can lead to serious problems such as failure of bone marrow stem cell transplantation, failure of hematopoietic reconstitution, infection, or relapse. Excessive restrictions on T cell removal will leave cells that are still capable of triggering GvHD. Therefore, current methods for treating GvHD are only successful in limited donor and host combinations, preventing many patients from receiving potentially life-saving treatments.

Mesenchymal stem cells (MSCs) display potent immunosuppressive activity and have been successfully used in clinical settings to treat graft-versus-host disease (GvHD), an otherwise lethal complication of bone marrow transplantation. Due to limited characterization, MSC preparations are quite heterogeneous and this limits the extent of their immunosuppressive effects and, therefore, their clinical benefit.

Overview

In the work leading to the present invention, the inventors compared the effects of mesenchymal stem cells and STRO-1 ^bright pluripotent cell preparations on GvHD. Surprisingly, the STRO-1 ^bright pluripotent cell preparation was far superior to the mesenchymal stem cell preparation in improving GvHD.

Thus, the present invention provides a method for reducing the development of GvHD complications in a mammalian patient, the method comprising administering to the patient a cell population enriched for STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny.

In one embodiment, the mammalian patient is undergoing or about to undergo a bone marrow transplant.

In another embodiment, the present invention provides a method for reducing the development of GvHD complications caused by bone marrow transplantation in a mammalian patient, the method comprising administering to the patient (a) precursors of myeloid lineage cells and (b) a cell population enriched in STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny, wherein the cell population enriched in STRO-1 ^bright cells and/or their progeny and/or the soluble factors obtained from the STRO-1 ^bright cells and/or their progeny are administered in an amount effective to reduce the severity of the patient's GvHD.

In one embodiment, graft-versus-host disease is caused by a T cell immune response. In one example, the T cells are from the donor and the antigen is from the recipient. For example, the T cells can be present in the transplant. In another embodiment, the T cells are from the recipient and the antigen is from the donor.

In another embodiment of this method, the Stro-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from said Stro-1 ^bright cells and/or their progeny cells are genetically engineered to express a molecule that blocks co-stimulation of T cells.

The STRO-1 ^bright cells can be autologous or allogeneic. In one embodiment, the STRO-1 ^bright cells are allogeneic.

In another embodiment of this method, the STRO-1 ^bright cells and/or progeny cells thereof have been expanded in culture prior to administering or obtaining the soluble factors.

Exemplary dosages of cells include between 0.1×10 ⁶ and 5×10 ⁶ STRO-1 ^bright cells and/or their progeny. For example, the method includes administering between 0.3×10 ⁶ and 2×10 ⁶ STRO-1 ^bright cells and/or their progeny.

One form of the method involves administering a low dose of STRO-1 ^bright cells and/or their progeny. Such a low dose is, for example, between 0.1×10 ⁵ and 0.5×10 ⁶ STRO-1 ^bright cells and/or their progeny, such as about 0.3×10 ⁶ STRO-1 ^bright cells and/or their progeny.

The present invention also encompasses multiple administrations of the cells and/or soluble factors. For example, the method may involve administering the cells and monitoring the subject to determine when one or more symptoms of GvHD develop or recur, and administering another dose of the cells and/or soluble factors. Suitable methods for assessing symptoms of GvHD will be apparent to those skilled in the art and/or are described herein.

In one example, the population enriched for STRO-1 ^bright cells and/or progeny thereof and/or soluble factors derived from said STRO-1 ^bright cells and/or progeny thereof are administered once a week or less frequently, such as once every four weeks or less frequently.

In another embodiment, the cell population enriched for STRO-1 ^bright cells and/or their progeny cells and/or the soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells are systemically administered. For example, the cell population enriched for STRO-1 ^bright cells and/or their progeny cells and/or the soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells can be administered intravenously, intraarterially, intramuscularly, subcutaneously, into the aorta, into the atria or ventricles of the heart, or into blood vessels connected to organs, such as the abdominal aorta, superior mesenteric artery, pancreaticoduodenal artery, or splenic artery.

In another embodiment, the method of the present invention further comprises administering an immunosuppressant. The immunosuppressant may be administered for a period of time sufficient to allow the transplanted cells to function. In one example, the immunosuppressant is cyclosporine. Cyclosporine may be administered at a dose of 5 mg to 40 mg per kilogram of body weight.

In this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step or group of elements, integers or steps but not the exclusion of any other element, integer or step or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Co-expression of TNAP (STRO-3) and the mesenchymal precursor cell marker STRO-1 ^bright in adult BMMNCs. Two-color immunofluorescence and flow cytometry were performed by incubating MACS-selected BMMNCs with STRO-1 indirectly labeled with goat anti-mouse IgM antibody conjugated to FITC (x-axis) and STRO-3 mAb indirectly labeled with goat anti-mouse IgG conjugated to PE (murine IgG1) (y-axis). The dot matrix histogram represents 5× ¹⁰⁴ events collected in list mode data format. The vertical and horizontal lines are set at a reactivity level of less than 1.0% of the mean fluorescence obtained with isotype-matched control antibodies 1B5 (IgG) and 1A6.12 (IgM) treated under the same conditions. The results show that a small population of STRO-1 ^bright cells co-expresses TNAP (upper right quadrant), while the remaining STRO-1+ cells fail to react with STRO-3 mAb.

Figure 2. Gene expression profiles of STRO-1 ^bright or STRO-1 ^dark progeny of cultured and expanded STRO-1 ^bright MPCs. Single cell suspensions of ex vivo expanded bone marrow MPCs were prepared by trypsin/EDTA treatment. Cells were stained with STRO-1 antibodies, which were then visualized by incubation with goat anti-mouse IgM-fluorescein isothiocyanate. After fluorescence activated cell sorting (A), total cellular RNA was prepared from the purified populations of cells expressing STRO-1 ^dark or STRO-1 ^bright . Total RNA was isolated from each subpopulation using the RNAzolB extraction method and standard procedures and used as a template for cDNA synthesis. The expression of various transcripts was assessed by PCR amplification using a standard protocol as previously described (Gronthos et al. J Cell Sci. 116: 1827-1835, 2003). The primer sets used in this study are shown in Table 2. After amplification, each reaction mixture was analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining (B). ImageQant software was used to assess the relative gene expression of each cell marker with reference to the expression of the housekeeping gene GAPDH (C).

Figure 3. STRO-1 ^bright progeny of cultured and amplified STRO-1 ⁺ MPCs express high levels of SDF-1, whereas STRO-1 ^dark progeny do not. (A) STRO-1 ⁺ BMMNC preparations separated by MACS were divided into different STRO-1 subsets based on the regions STRO-1 ^bright and STRO-1 ^dark/dark using FACS. Total RNA was prepared from each STRO-1 subpopulation and used to construct STRO-1 ^bright subtractive hybridization libraries (BC). Nitrocellulose filters were replicated and dotted with representative PCR products amplified from bacterial clones transformed with STRO-1 ^bright subtractive cDNA. The filters were then probed with [ ³² P] deoxycytidine triphosphate (dCTP)-labeled STRO-1 ^bright (B) or STRO-1 ^dark/dark (C) subtracted cDNA. Arrows indicate differential expression of one clone containing a cDNA fragment corresponding to human SDF-1. (D) Reverse transcriptase (RT)-PCR analysis showing the relative expression of SDF-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in total RNA prepared from freshly isolated BMMNC STRO-1 populations by MACS/FACS before culture. bp indicates base pairs.

Figure 4. Comparison of the efficacy of STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) in inhibiting T cell proliferation. PBMCs were stimulated with CD3/CD28-coated beads for 4 days in the absence or presence of Preparations A or B. T cell proliferation was measured by counts per minute (cpm) via ^3H -Tdr incorporation.

Figure 5. Comparison of the efficacy of STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) in inhibiting T cell proliferation. PBMCs were stimulated with CD3/CD28-coated beads for 4 days in the presence of varying concentrations of Preparation A or B. T cell proliferation in various cultures was measured by ^3H -Tdr incorporation and reported as a percentage of control T cell proliferation when PBMCs were stimulated in the absence of MSCs.

Figure 6. Comparison of the effects of STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) on GvHD. Bone marrow mononuclear cells (BMMCs) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, the recipient mice were injected with 1×10 ⁶ MSCs (Preparation A1) or MPCs (Preparation B1) per mouse or the recipient mice did not receive further treatment (control). Eight mice per group were injected. The mice were evaluated at one-week intervals. Time represents the number of weeks.

Figure 7. Comparison of the effects of STRO-1 negative MSCs (preparation A) and STRO-1 ^bright MPCs (preparation B) on GvHD. Bone marrow mononuclear cells (BMMCs) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, the recipient mice were injected with 1 or 2×10 ⁶ cells of preparations A or B (A1, A2, B1, B2), or the recipient mice did not receive further treatment (control). Eight mice per group were injected.

Figure 8. Comparison of the effects of high-dose STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) on GvHD. Bone marrow mononuclear cells (BMMCs) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. Four weeks later, the recipient mice were injected with 2×10 ⁶ cells of preparations A or B (A2, B2), or the recipient mice did not receive further treatment (Control 2). Eight mice per group were injected, but 4 and 3 mice in groups B2 and A2, respectively, died shortly after injection.

Figure 9. Comparison of the effects of low-dose STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) on GvHD. Bone marrow mononuclear cells (BMMCs) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, the recipient mice were injected with 0.3×10 ⁶ cells of Preparation A or B (A0.3, B0.3), or the recipient mice did not receive further treatment (Control 2). Six mice per group were injected. The figure reports the GvHD score for each mouse.

Figure 10. Comparison of the effects of low-dose STRO-1 negative MSCs (Preparation A) and STRO-1 ^bright MPCs (Preparation B) on GvHD. Bone marrow mononuclear cells (BMMCs) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, the recipient mice were injected with 0.3×10 ⁶ cells of Preparation A or B (A0.3, B0.3), or the recipient mice did not receive further treatment (Control 2). Six mice per group were injected. The figure reports the average GvHD score for each mouse.

Detailed description

The results presented herein demonstrate that cell populations enriched for STRO-1 ^bright cells are unexpectedly superior to STRO-1 negative mesenchymal stem cell preparations in ameliorating GvHD.

Thus, the present invention provides a method for reducing the development of GvHD complications in a mammalian patient, the method comprising administering to the patient a cell population enriched for STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny.

For example, the present invention provides a method for reducing the development of GvHD complications caused by bone marrow transplantation in a mammalian patient, the method comprising administering to the patient (a) precursors of bone marrow lineage cells and (b) a cell population enriched in STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny, wherein the cell population enriched in STRO-1 ^bright cells and/or their progeny and/or the soluble factors obtained from the STRO-1 ^bright cells and/or their progeny are administered in an amount effective to reduce the severity of the patient's GvHD.

As used herein, the term "soluble factor" is understood to refer to any molecule produced by STRO-1 ^bright cells and/or their progeny that is soluble in water, such as proteins, peptides, glycoproteins, glycopeptides, lipoproteins, lipopeptides, carbohydrates, etc. The soluble factors can be intracellular and/or secreted by the cells. The soluble factors can be a complex mixture (e.g., a supernatant) and/or its components and/or can be purified factors. In one embodiment of the present invention, the soluble factors are supernatants or are contained in supernatants. Therefore, any embodiment herein involving the administration of one or more soluble factors should be understood as necessary modifications to the administration of the supernatant.

The methods of the present invention may involve administering a cell population enriched solely for STRO-1- ^bright cells and/or their progeny cells, and/or soluble factors derived from said STRO-1- ^bright cells and/or their progeny cells. The methods of the present invention may also involve administering solely progeny cells, or soluble factors derived from said progeny cells. The methods of the present invention may also involve administering a mixed population of STRO-1- ^bright cells and their progeny cells, or soluble factors derived from a mixed culture of STRO-1- ^bright cells and their progeny cells.

The present invention is preferably applied to humans, however, it is contemplated that the present invention is also applicable to animals, and these may include agricultural animals (such as cattle, sheep, pigs, etc.), domestic animals (such as dogs, cats), laboratory animals (such as mice, rats, hamsters and rabbits), or animals that can be used for sports (such as horses).

Therefore, STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells can be used to adapt the recipient's immune system to the donor or foreign bone marrow cells by administering to the recipient an amount of STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells that effectively reduces or eliminates the recipient's T cell immune response against the transplant before or simultaneously with the transplantation of the donor cells. STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells affect the recipient's T cells so that the T cell response is reduced or eliminated when the donor or foreign tissue is present.

Therefore, in the case of bone marrow (hematopoietic stem cell) transplantation, the attack of the graft on the host can be reduced or eliminated. Before the bone marrow or peripheral blood stem cells are transplanted into the recipient, the donor bone marrow can be pretreated with the recipient's STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells. In a preferred embodiment, the donor bone marrow is first exposed to the recipient's tissue/cells and then treated with STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells. Although not limited, it is believed that the effect of the initial contact with the recipient's tissue or cells is to activate T cells in the bone marrow. Subsequent treatment with STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells inhibits or eliminates further activation of T cells in the bone marrow, thereby reducing or eliminating the adverse effects of the donor tissue, that is, the therapy reduces or eliminates the graft-versus-host reaction.

In another embodiment, a transplant recipient suffering from graft-versus-host disease can be treated to reduce or eliminate the severity of the disease by administering to the recipient an amount of the donor's own or allogeneic STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells, in an amount effective to reduce or eliminate the host's graft rejection reaction. The allogeneic cells can be STRO-1 ^bright cells and/or their progeny cells that are allogeneic to the recipient or third-party STRO-1 ^bright cells and/or their progeny cells. STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells inhibit or suppress activated T cells in the donor tissue from initiating an immune response against the recipient, thereby reducing or eliminating the graft-versus-host reaction.

The recipient's STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells can be obtained from the recipient before transplantation and can be stored and/or cultured and expanded to provide a reserve pool of STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells in an amount sufficient to treat ongoing graft-versus-host attack.

It is further contemplated that only a single treatment with the STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells of the present invention may be required, thereby eliminating the need for long-term immunosuppressive drug therapy. Alternatively, multiple administrations of STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells may be used.

The dosage of STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from said STRO-1 ^bright cells and/or their progeny cells can vary within wide limits and will, of course, be adapted to the individual requirements of each particular case. Generally speaking, in the case of parenteral administration, about 10,000 to 5 million cells are usually administered per kilogram of recipient body weight. The number of cells used will depend on the weight and condition of the recipient, the number or frequency of administration, and other variables known to those skilled in the art.

The cells can be suspended in an appropriate diluent at a concentration of about 0.01 to about 5 x 10 ⁶ cells per milliliter. One form of the method involves administering a low dose of STRO-1 ^bright cells and/or their progeny. Such a low dose is, for example, between 0.1 x 10 ⁵ and 0.5 x 10 ⁶ STRO-1 ^bright cells and/or their progeny, such as about 0.3 x 10 ⁶ STRO-1 ^bright cells and/or their progeny.

Suitable excipients for injectable solutions are those that are biologically and physiologically compatible with the cells and with the recipient, such as buffered saline solutions or other suitable excipients. Compositions for administration are preferably formulated, prepared, and stored according to standard methods consistent with appropriate sterility and stability.

The following examples further illustrate various aspects of the present invention. However, they in no way limit the teachings and disclosures of the present invention as described herein.

STRO-1 ^bright cells or progeny cells, and supernatant or one or more soluble factors obtained therefrom

STRO-1 ^bright cells are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, bones, ligaments, tendons, skeletal muscle, dermis and periosteum; and are generally capable of differentiating into the germ line, such as mesoderm and/or endoderm and/or ectoderm. Therefore, STRO-1 ^bright cells are capable of differentiating into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage determination and differentiation path entered by these cells depends on various influences from mechanical influences and/or endogenous bioactive factors (such as growth factors, cytokines) and/or local microenvironmental conditions established by the host tissue. STRO-1 ^bright cells are therefore preferably non-hematopoietic progenitor cells that divide to produce daughter cells, which are stem cells or precursor cells that irreversibly differentiate to produce phenotypic cells in due time.

In a preferred embodiment, STRO-1 ^bright cells are enriched from a sample obtained from a subject, e.g., the subject to be treated, a related subject, or an unrelated subject (whether of the same species or a different species). The term "enriched" or variations thereof is used herein to describe a cell population in which the proportion of a particular cell type or the proportions of multiple particular cell types are increased when compared to an untreated population.

In one embodiment, STRO-1 ^bright cells are preferentially enriched relative to STRO-1 ^dim or STRO-1 ^intermediate cells.

In a preferred embodiment, the cells used in the present invention express one or more markers individually or collectively selected from the group consisting of TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or any combination thereof.

"Individually" means that the present invention independently encompasses the marker or marker group, and that even though an individual marker or marker group may not be individually listed herein, the appended claims may still define the marker or marker group independently and separately from each other.

"Encyclopedia" means that the present invention covers any number of the aforementioned markers or peptide groups or combinations thereof, and although the aforementioned number of markers or marker groups or combinations thereof cannot be specifically listed herein, the appended claims may still define the aforementioned combination or subcombination independently and separately from any other combination of markers or marker groups.

Preferably, the STRO-1 ^bright cells are additionally one or more of TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ and/or CD146 ⁺ .

A cell that is designated "positive" for a given marker may express low (lo or dark) or high (bright, bright) levels of that marker, depending on the extent to which the marker is present on the cell surface, wherein the terms relate to the intensity of the fluorescent or other marker used in the cell sorting process. The distinction between lo (or dark or deep) and bright should be understood in the context of the marker used on the specific cell population being sorted. A cell that is designated "negative" for a given marker is not necessarily completely absent from the cell. The term refers to a cell that expresses the marker at a relatively low level and, when detectably labeled, produces a very low signal or is undetectable above background levels.

As used herein, the term "bright" refers to a marker on the cell surface that produces a relatively high signal when detectably labeled. While not wishing to be bound by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., an antigen recognized by STRO-1) than other cells in the sample. For example, when labeled with a FITC-conjugated STRO-1 antibody, STRO-1 ^bright cells produce a higher fluorescent signal than non-bright cells ((STRO-1 ^dark/dark )) as determined by fluorescence activated cell sorting (FACS) analysis. Preferably, the "bright" cells constitute at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, the "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In a preferred embodiment, the STRO-1 ^bright cells have a STRO-1 surface expression of more than 2 logs relative to "background" (i.e., STRO-1 ^- cells). In contrast, the STRO-1 ^dark and/or STRO-1 ^intermediate cells have a STRO-1 surface expression of less than 2 logs relative to "background," typically less than about 1 log relative to "background."

As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses the hepatic isoform (LAP), the bone isoform (BAP), and the renal isoform (KAP). In a preferred embodiment, the TNAP is BAP. In a particularly preferred embodiment, the TNAP as used herein refers to a molecule that can bind to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005, under the provisions of the Budapest Treaty under deposit number PTA-7282.

Furthermore, in a preferred embodiment, the STRO-1 ^bright cells are capable of producing clonal CFU-F.

It is preferred that a large proportion of multipotent cells can differentiate into at least two different germ lines. The non-limiting examples of the pedigree that multipotent cells can develop into include bone precursor cells; Hepatocyte progenitor cells, which have the multipotency of biliary epithelial cells and hepatocytes; Neural restricted cells, which can produce glial cell precursors that develop into oligodendrocytes and astrocytes; Neuronal precursors, which can develop neurons; Myocardial and myocardial cells, glucose-responsive insulin secreting pancreatic beta cell line precursors. Other pedigrees include but are not limited to odontoblasts, dentin-producing cells and chondrocytes, and following precursor cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, ureteral epithelial cells, smooth muscle and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, myocardial, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glial cells, neurons, astrocytes and oligodendrocytes.

In another embodiment, the STRO-1 ^bright cells are unable to generate hematopoietic cells in culture.

In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques and used to obtain supernatant or soluble factors or expanded cells for administration to the subject in the form of an autologous or allogeneic composition. In an alternative embodiment, cells from one or more established human cell lines are used. In another applicable embodiment of the present invention, cells from a non-human animal (or from another species if the patient is not human) are used.

The present invention also encompasses the use of supernatants or soluble factors obtained or obtained from STRO-1 ^bright cells and/or their progeny cells (the latter also referred to as expanded cells) produced from in vitro cultures. The expanded cells of the present invention may have a variety of phenotypes depending on the culture conditions (including the amount and/or type of stimulating factors in the culture medium), the number of passages, and the like. In certain embodiments, the progeny cells are obtained after being passaged from the parent population for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times. However, the progeny cells may be obtained after any number of passages from the parent population.

Daughter cells can be obtained by cultivating in any suitable culture medium. As used herein, the term "culture medium" includes components of the cell surrounding environment. Culture medium can be a mixture of solid, liquid, gaseous or phase and material. Culture medium includes liquid growth medium and liquid culture medium that does not support cell growth. Culture medium also includes colloidal culture medium, such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous culture medium includes the gas phase exposed to cells grown on a petri dish (petri dish) or other solid or semi-solid support. The term "culture medium" also refers to the material intended for cell culture, even if it has not yet contacted with cells. In other words, the nutrient-rich liquid prepared for bacterial culture is culture medium. The powder mixture that becomes suitable for cell culture when mixed with water or other liquids can be called "powdered culture medium".

In one embodiment, progeny cells suitable for use in the methods of the present invention are obtained by isolating TNAP ⁺ STRO-1 ⁺ multipotent cells from bone marrow using magnetic beads labeled with STRO-3 antibodies, and then culturing and expanding the isolated cells (for examples of suitable culture conditions, see Gronthos et al., Blood 85:929-940, 1995).

In one embodiment, the expanded cells (progeny) (preferably after at least 5 passages) may be ^TNAP- , CC9 ⁺ , HLA class I ⁺ , HLA class II- ^{, CD14-} , ^CD19- , CD3-, CD11a ^{- c-} , ^{CD31-, CD86- , CD34- ,} and/or ^CD80- . However, under culture conditions different from those described herein, the expression of different markers may vary. In addition, although cells with these phenotypes may predominate in the expanded cell population, this does not mean that a smaller proportion of cells do not have this phenotype (e.g., a small percentage of expanded cells may be ^CC9- ). In a preferred embodiment, the expanded cells still have the ability to differentiate into different cell types.

In one embodiment, the expanded cell population used to obtain the supernatant or soluble factors or the cells themselves comprises cells wherein at least 25%, more preferably at least 50% of the cells are CC9 ⁺ .

In another embodiment, the expanded cell population used to obtain the supernatant or soluble factors or the cells themselves comprises cells in which at least 40%, more preferably at least 45%, of the cells are STRO-1 ⁺ .

In another embodiment, the expanded cells may express one or more markers collectively or individually selected from the group consisting of LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD90, CD29, CD18, CD61, integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2 = leptin-R), RANKL, STRO-1 ^beta and CD146, or any combination of these markers.

In one embodiment, the daughter cells derived from STRO-1 ^bright cells are positive for the marker Stro-1 ^dark . These cells are called tissue-specific committed cells (TSCC) and are more prone to differentiation than STRO-1 ^bright cells and therefore have a lower ability to respond to inducing factors. Non-limiting examples of lineages that TSCC can develop into include hepatocyte progenitor cells, which have the multipotency of biliary epithelial cells and hepatocytes; neural restricted cells, which can produce glial cell precursors that develop into oligodendrocytes and astrocytes; and neuronal precursors, which develop into neurons; myocardium and myocardial cells, glucose-responsive insulin-secreting pancreatic beta cell lineage precursors. Other directed lineages include, but are not limited to, chondrocytes, osteoblasts, odontoblasts, tooth-producing cells and cartilage cells, and following precursor cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, ureteral epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, osteoclasts and hematopoietic support matrix, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glial cells, neurons, astrocytes and oligodendrocytes. Precursors include those cells that clearly can produce connective tissue, particularly including adipose tissue, alveolar tissue, bone tissue, cartilage tissue, elastic tissue and fibrous connective tissue.

In another embodiment, the progeny cells are multipotent expanded STRO-1 ⁺ multipotent cell progeny (MEMP) as defined and/or described in WO 2006/032092. Methods for preparing enriched populations of STRO-1 ⁺ multipotent cells that can be used to obtain progeny are described in WO 01/04268 and WO 2004/085630. In an in vitro situation, STRO-1 ⁺ multipotent cells rarely exist in the form of an absolutely pure preparation, but are generally present together with other cells that are tissue-specific committed cells (TSCCs). WO01/04268 proposes to collect the cells from the bone marrow at a purity level of about 0.1% to 90%. The population containing MPCs that can obtain progeny can be collected directly from a tissue source, or it can be a population that has been expanded ex vivo.

For example, progeny can be obtained from a harvested and unexpanded population of substantially purified STRO-1 ⁺ pluripotent cells that constitute at least about 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 95% of the total cells of the population in which they are present. This level can be achieved, for example, by selecting cells that are positive for at least one marker, individually or collectively, selected from the group consisting of TNAP, STRO-1 ^bright , 3G5 ⁺ , VCAM-1, THY-1, CD146, and STRO-2.

MEMPS can be distinguished from freshly collected STRO-1 bright cells in that they are positive for the marker STRO-1 ^bright and negative for the marker alkaline phosphatase (ALP). In contrast, freshly isolated Stro-1 ^bright cells are positive for both STRO-1 ^bright and ALP. In a preferred embodiment of the present invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have the phenotype STRO-1 ^bright , ALP ^- . In another preferred embodiment, MEMPS are positive for one or more of the markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In another preferred embodiment, MEMPs do not show TERT activity and/or are negative for the marker CD18.

The starting population of STRO-1 ^bright cells can be derived from any one or more tissue types, including bone marrow, dental pulp cells, adipose tissue, and skin, and perhaps more broadly from adipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles, intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament, bone marrow, tendon, and skeletal muscle.

It will be appreciated that in practicing the present invention, the isolation of cells bearing any given cell surface marker can be achieved by a variety of different methods, however, preferred methods rely on the binding of a binding agent (e.g., an antibody or antigen-binding fragment thereof) to the marker of interest, followed by separation of those cells that exhibit binding (either high or low levels of binding) or no binding. The most suitable binding agent is an antibody or an antibody-based molecule, preferably a monoclonal antibody or a monoclonal antibody-based molecule, because the latter have specificity. Antibodies can be used in both steps, however, other reagents can also be used, and thus ligands for these markers can also be used to enrich for cells bearing or lacking the marker.

The antibody or ligand can be attached to a solid support to allow for a rough separation. The separation technique preferably maintains the maximum viability of the portion to be collected. Various techniques with different efficacies can be used to obtain a relatively rough separation. The specific technique used will depend on the separation efficiency, associated cytotoxicity, ease and speed of execution, and the necessity of complex equipment and/or technical skills. Separation procedures may include, but are not limited to, magnetic separation using magnetic beads coated with antibodies, affinity chromatography, and "panning" utilizing antibodies attached to a solid matrix. Technologies providing precise separation include, but are not limited to, FACS. The method for implementing FACS is apparent to those skilled in the art.

Antibodies to each of the markers described herein are commercially available (eg, monoclonal antibodies against STRO-1 are commercially available from R&D Systems, USA), obtained from the ATCC or other depository organizations, and/or can be generated using art-recognized techniques.

The method for isolating STRO-1 ^bright cells preferably includes, for example, a first step, which is a solid phase sorting step using, for example, magnetic activated cell sorting (MACS) that identifies high levels of STRO-1 expression. This can be followed, if necessary, by a second sorting step to generate even higher levels of precursor cell expression. This second sorting step may involve the use of two or more markers.

Methods for obtaining STRO-1- ^positive cells may also include harvesting the source cells prior to the first enrichment step using known techniques. Thus, the tissue is surgically removed. The cells comprising the source tissue are then isolated into a so-called single-cell suspension. This isolation can be achieved physically or enzymatically.

Once a suitable population of STRO-1 ^bright cells is obtained, they can be cultured or expanded by any suitable means to obtain MEMPs.

In one embodiment, cells are taken from the subject to be treated, cultured in vitro using standard techniques and used to obtain supernatants or soluble factors or expand cells for administration to the subject as an autologous or allogeneic composition. In an alternative embodiment, cells from one or more established human cell lines are used to obtain supernatants or soluble factors. In another applicable embodiment of the invention, cells from non-human animals (or from another species if the patient is not human) are used to obtain supernatants or soluble factors.

The present invention can be practiced using cells from any non-human animal species, including but not limited to non-human primate cells, ungulates, canines, felines, lagomorphs, rodents, avians, and fish. Primate cells useful in practicing the present invention include, but are not limited to, cells from chimpanzees, baboons, cynomolgus macaques, and any other New World or Old World monkeys. Ungulates useful in practicing the present invention include, but are not limited to, cells from cattle, pigs, sheep, goats, horses, buffalo, and bison. Rodents useful in practicing the present invention include, but are not limited to, cells from mice, rats, guinea pigs, hamsters, and gerbils. Examples of lagomorph species useful in practicing the present invention include rabbits, jackrabbits, hares, cottontail rabbits, snow hares, and pikas. Chickens (Gallus gallus) are an example of avian species useful in practicing the present invention.

Cells suitable for use in the methods of the present invention may be stored prior to use or prior to obtaining supernatants or soluble factors. Methods and protocols for preserving and storing eukaryotic cells, and in particular mammalian cells, are known in the art (see, for example, Pollard, J.W. and Walker, J.M. (1997) Basic Cell Culture Protocols, 2nd ed., Humana Press, Totowa, N.J.; Freshney, R.I. (2000) Culture of Animal Cells, 4th ed., Wiley-Liss, Hoboken, N.J.).

Genetically modified cells

In one embodiment, STRO-1 ^cells and/or progeny cells thereof are genetically modified, e.g., to express and/or secrete a protein of interest, e.g., a protein that provides a therapeutic and/or prophylactic benefit, such as insulin, glucagon, somatostatin, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase, or amylase, or a polypeptide that is associated with enhanced angiogenesis or causes angiogenesis, or a polypeptide that is associated with cell differentiation into pancreatic cells or vascular cells.

The method for genetically modifying cells is apparent to those skilled in the art. For example, the nucleic acid for expression in the cell is operably connected to a promoter for inducing expression in the cell. For example, nucleic acid is connected to a promoter operable in the various cells of the experimenter, such as a viral promoter, for example a CMV promoter (for example a CMV-IE promoter) or a SV-40 promoter. Other suitable promoters are known in the art and are understood to be applicable to embodiments of the present invention mutatis mutandis.

Preferably, the nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid having the ability to confer expression to an operably linked nucleic acid (e.g., a reporter gene and/or a counterselectable reporter gene) in a cell. In the context of the present invention, it will be understood that an expression construct may comprise or be a plasmid, phage, phagemid, cosmid, viral subgenome or genomic fragment or other nucleic acid capable of maintaining and/or replicating heterologous DNA in an expressible format.

Methods for constructing expression constructs suitable for practicing the present invention will be readily apparent to those skilled in the art and are described in, for example, Ausubel et al. (Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al. (Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 3rd edition 2001). For example, the components of the expression construct are amplified from a suitable template nucleic acid using, for example, PCR and subsequently cloned into a suitable expression construct, such as a plasmid or phagemid.

Suitable vectors for the expression constructs are known in the art and/or described herein. For example, expression vectors suitable for the methods of the invention in mammalian cells are, for example, vectors of the pcDNA vector suite, the pCI vector suite (Promega), the pCMV vector suite (Clontech), the pM vector (Clontech), the pSI vector (Promega), the VP 16 vector (Clontech) or the pcDNA vector suite (Invitrogen) supplied by Invitrogen.

Those skilled in the art will recognize other vectors and sources of such vectors, such as Invitrogen, Clontech, or Promega.

The mode for expressing for the nucleic acid molecule of separation or the gene construct that comprises described nucleic acid is introduced into the cell is known to those skilled in the art.The technology for specifying organism depends on the technology of known success.The mode for recombinant DNA to be introduced into the cell comprises microinjection, DEAE-dextran-mediated transfection, liposome (such as using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA)) mediated transfection, DNA absorption, electroporation and microparticle bombardment (such as using tungsten particles or gold particles (Agracetus Inc., WI, USA) and other mode that are coated with DNA) that are used.

Alternatively, the expression construct of the present invention is a viral vector. Suitable viruses are known in the art and are commercially available. Conventional viral-based systems for delivering nucleic acids and integrating the nucleic acids into the host cell genome include, for example, retroviral vectors, lentiviral vectors, or adeno-associated viral vectors. Alternatively, adenoviral vectors are suitable for introducing nucleic acids into host cells in a free form. Viral vectors are an effective and versatile method for gene transfer in target cells and tissues. In addition, high transduction efficiencies have been observed in a variety of different cell types and target tissues.

For example, retroviruses typically contain cis-acting long terminal repeats (LTRs) with the capacity to package up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the expression construct into target cells to provide long-term expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739 (1992); Johann et al., J. Virol. 65:1635-1640 (1992); Sommerfelt et al., Virol. 76:58-59 (1990); Wilson et al., J. Virol. 63:274-2318 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller and Rosman BioTechniques 7:980-990, 1989; Miller, A.D. Human Gene Therapy 7:5-14, 1990; Scarpa et al. Virology 75:849-852, 1991; Burns et al. Proc. Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have been developed for nucleic acid delivery. AAV vectors can be readily constructed using techniques known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka. Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, Human Gene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169, 1994; and Zhou et al. J. Exp. Med. 179:1867-1875,1994.

Other viral vectors suitable for delivery of the expression constructs of the invention include, for example, vectors derived from the poxvirus family, such as vaccinia virus and fowlpox virus, or alphavirus or conjugate viral vectors (e.g., those described in Fisher-Hoch et al., Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Analysis of the therapeutic/preventive potential of cellular and soluble factors

Methods for determining the ability of soluble factors derived from STRO-1 ^bright cells to treat or prevent GvHD or delay the onset or development of GvHD will be apparent to those skilled in the art.

For example, an in vitro assay suitable for determining the immunosuppressive activity of soluble factors is described in Example 5 herein.

In another example, the efficacy of soluble factors can be assessed in an in vivo model of GvHD as described in Examples 6 and 7 herein.

It will be apparent to those skilled in the art based on the foregoing that the present invention also provides a method for identifying or isolating soluble factors for treating, preventing or delaying GvHD, the method comprising:

(i) administering a soluble factor to a test subject suffering from GvHD and assessing the progression of GvHD in the subject;

(ii) comparing the level of GvHD in the subject of (i) with the level of GvHD in a control subject who has GvHD but has not been administered the soluble factor,

Wherein GvHD is reduced in the test subject compared to the control subject, it indicates that the soluble factor treats, prevents or delays GvHD.

Cell composition

In one embodiment of the present invention, STRO-1 ^bright cells and/or progeny cells thereof are administered in the form of a composition. Preferably, the composition comprises a pharmaceutically acceptable carrier and/or excipient.

The terms "carrier" and "excipient" refer to compositions of matter routinely used in the art to facilitate the storage, administration, and/or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mac Publishing Company (1980). A carrier can also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., unable to react with other components of the carrier. In one example, the carrier does not produce significant local or systemic adverse effects in the recipient at the dosages and concentrations used for treatment.

Suitable carriers for the present invention include those commonly used carriers, such as water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronic acid and glycols are preferred liquid carriers, especially (when isotonic) solution carriers. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

In another example, the carrier is a medium composition, such as a medium composition that cells are grown or suspended in. Preferably, the medium composition does not induce any adverse effects in a subject to which it is administered.

Preferably, the vehicle and excipients do not adversely affect the viability of the cells and/or the ability of the cells to reduce, prevent or delay pancreatic dysfunction.

In one example, the carrier and excipient provide buffering activity to maintain cells and/or soluble factors at a suitable pH, thereby exerting biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows for rapid release of cells and factors, in which case the compositions of the invention can be manufactured as a liquid for direct application, for example, by injection, to the bloodstream or tissue or to an area surrounding or adjacent to the tissue.

STRO-1 ^cells and/or their progeny cells can also be incorporated into or embedded in a scaffold that is compatible with the recipient and degrades into products that are harmless to the recipient. Such a scaffold provides support and protection for the cells to be transplanted into the recipient. Natural and/or synthetic biodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds can be successfully used in the practice of the present invention. Preferred scaffolds include, but are not limited to, biodegradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Synthetic materials suitable for cell transplant scaffolds should be able to support a wide range of cell growth and cell function. The scaffolds are resorbable. Suitable scaffolds include polyglycolic acid scaffolds, such as those described in Vacanti et al., J. Ped. Surg. 23:3-9 1988; Cima et al., Biotechnol. Bioeng. 38:145 1991; Vacanti et al., Plast. Reconstr. Surg. 88:753-9 1991, or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

In another example, cells can be administered in a gel scaffold such as Gelfoam from Upjohn.

Cell compositions suitable for use in the present invention can be administered alone or as a mixture with other cells. Cells that can be administered in conjunction with the compositions of the present invention include, but are not limited to, other multipotent or pluripotent cells or stem cells, or bone marrow cells. Different types of cells can be mixed with the compositions of the present invention immediately before or shortly before administration, or they can be co-cultured together for a period of time before administration.

Preferably, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about 1×10 ⁵ STRO-1 ^bright cells/kg to about 1×10 ⁷ STRO-1 ^bright cells/kg or about 1×10 ⁶ STRO-1 ^bright cells/kg to about 5×10 ⁶ STRO-1 ^bright cells/kg. The exact amount of cells to be administered depends on a variety of factors, including the patient's age, weight, and sex, as well as the extent and severity of the pancreatic dysfunction.

In some embodiments, the cells are contained in a chamber that does not allow the cells to escape into the subject's circulation, but does allow factors secreted by the cells to enter the circulation. In this way, soluble factors can be administered to a subject by allowing the cells to secrete factors into the subject's circulation. The chamber can also be implanted in a location on a subject to increase local levels of soluble factors, such as in or near a transplanted organ.

In some embodiments of the present invention, it may not be necessary or desirable to immunosuppress the patient prior to initiating cell composition therapy.Thus, transplantation of allogeneic or even xenogeneic STRO-1 ^bright cells or their progeny may be tolerogenic in some circumstances.

However, in other cases, it may be necessary or appropriate to pharmacologically immunosuppress the patient before starting cell therapy. This can be achieved by using systemic or local immunosuppressants, or this can be achieved by delivering cells in an encapsulation device. The cells can be encapsulated in a capsule that is permeable to the nutrients and oxygen required by the cells and therapeutic factors, but impermeable to the cells to immunize humoral factors and cells. Preferably, the encapsulating agent has low allergenicity, is easily and stably located in the target tissue, and provides additional protection to the implanted structure. These and other ways to reduce or eliminate the immune response to transplanted cells are known in the art. Alternatively, the cells can be genetically modified to reduce their immunogenicity.

Composition of soluble factors

In one embodiment of the present invention, the supernatant or soluble factors obtained from STRO-1 ^cells and/or from progeny cells are administered in the form of a composition, for example, comprising a suitable carrier and/or excipient. Preferably, the carrier or excipient does not adversely affect the biological effects of the soluble factors or supernatant.

In one embodiment, the composition comprises a composition of matter that stabilizes soluble factors or supernatant components (eg, protease inhibitors). Preferably, the amount of protease inhibitor included is insufficient to adversely affect the subject.

Compositions comprising supernatants or soluble factors obtained from STRO-1 ^bright cells and/or obtained from daughter cells can be prepared as appropriate liquid suspensions, such as suspensions in culture medium or in a stable carrier or buffer solution (e.g., phosphate-buffered saline). Suitable carriers are described above. In another example, the suspension comprising supernatants or soluble factors obtained from Stro-1 ^bright cells and/or obtained from daughter cells is an oily suspension for injection. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. The suspension to be used for injection may also contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or polydextrose. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution.

Sterile injectable solutions can be prepared by incorporating the required amount of supernatant or soluble factors in an appropriate solvent with one or a combination of the above ingredients, as required, followed by filtered sterilization.

In general, dispersions are prepared by incorporating the supernatant or soluble factors into a sterile vehicle containing a basic dispersion medium or other desired ingredients from the ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof. According to alternative aspects of the invention, the supernatant or soluble factors may be formulated with one or more other compounds that enhance its solubility.

Other exemplary carriers or excipients are described, for example, in Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, it is preferred to include an isotonic agent (e.g., sugar), a polyol (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption (e.g., monostearate and gelatin) in the composition. In addition, soluble factors can be administered in a time-release formulation, for example, in a composition comprising a slow-release polymer. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid (PLG) can be used. Various methods for preparing such formulations are patented or generally known to those skilled in the art.

The supernatant or soluble factors may be administered in combination with, for example, an appropriate matrix that provides slow release of the soluble factors.

Mode of administration

The supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny can be surgically implanted, injected, delivered (e.g., by means of a catheter or syringe) or otherwise administered directly or indirectly to a site (e.g., an organ) requiring repair or enhancement or to the blood system of a subject.

Preferably, the supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny are delivered to the bloodstream of the subject. For example, the supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny are delivered parenterally. Exemplary routes of parenteral administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal, intraventricular, intracerebroventricular, and intrathecal. Preferably, the supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny are delivered intra-arterially to the aorta, the atria or ventricles of the heart or the blood vessels connected to the pancreas (e.g., the abdominal aorta, the superior mesenteric artery, the pancreaticoduodenal artery or the splenic artery).

In the case of cell delivery to the atria or ventricles of the heart, it is preferred that the cells be administered to the left atrium or ventricle to avoid complications that may arise from rapid delivery of cells to the lungs.

Preferably, supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 bright cells, or progeny thereof are injected into the delivery site, for example, using a syringe or through a catheter or central line.

The choice of administration regimen for a therapeutic formulation depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, and the immunogenicity of the entity. Preferably, the administration regimen maximizes the amount of therapeutic compound delivered to the patient consistent with an acceptable level of side effects. Thus, the amount of formulation delivered depends, in part, on the specific entity and the severity of the condition being treated.

In one embodiment, the supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny are delivered as a single bolus dose. Alternatively, the supernatant or soluble factors obtained from STRO-1 ^bright cells, STRO-1 ^bright cells or their progeny are administered by continuous infusion, or by doses spaced, for example, one day, one week or 1-7 times per week. A preferred dosage regimen is one that involves maximizing the dose or avoiding significant undue side effects. The total weekly dose depends on the type and activity of the compound used. The appropriate dose is determined by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. In general, the dose starts with an amount slightly less than the optimal dose and is increased in small increments thereafter until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic indicators include diabetic symptom indicators.

Example

Example 1: MSC preparation

MSC is to be regenerated by bone marrow as described in US 5,837,539.About 80-100ml bone marrow is aspirated into the sterile syringe containing heparin and sent to MDACC Cell Therapy Laboratory (MDACC Cell Therapy Laboratory) for producing MSC. Use ficoll-hypaque to separate bone marrow mononuclear cells and be placed in two T175 flasks, wherein each flask has 50ml MSC expansion culture medium, and described culture medium comprises the α modified MEM (αMEM) containing gentamicin, glutamine (2mM) and 20% (v/v) fetal bovine serum (FBS) (Hyclone).

The cells were cultured at 37 RC, 5% CO ₂ for 2-3 days, at which time non-adherent cells were removed; the remaining adherent cells were cultured until the cell confluence reached 70% or higher (7-10 days), and then the cells were trypsinized and placed in six T175 flasks with MSC expansion medium (50 ml medium per flask). As described in Table 5 of US 5,837,539, MSCs isolated and expanded in this manner were STRO-1 negative.

Example 2: Immunoselection of MPCs by selecting STRO-3+ cells

Bone marrow (BM) was collected from healthy normal adult volunteers (20-35 years old) according to procedures approved by the Institutional Ethics Committee of the Royal Adelaide Hospital. Briefly, 40 ml of BM was aspirated from the posterior iliac crest into tubes containing lithium heparin anticoagulant.

BMMNCs were prepared by density gradient separation using Lymphoprep (Zannettino, AC et al. (1998) Blood 92:2613-2628) (Nycomed Pharma, Oslo, Norway) as ^previously described. After centrifugation at 400 × g for 30 minutes at 4 RC, the buffy coat was removed with a transfer pipette and washed three times in "HHF" consisting of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS; CSL Limited, Victoria, Australia).

STRO-3 ⁺ (or TNAP ⁺ ) cells were then isolated by magnetic-activated cell sorting as previously described (Gronthos et al. (2003) Journal of Cell Science 116:1827-1835; Gronthos, S. and Simmons, PJ (1995) Blood 85:929-940). Briefly, approximately 1-3 × ¹⁰⁸ BMMNCs were incubated on ice for 20 minutes in blocking buffer consisting of 10% (v/v) normal rabbit serum in HHF. The cells were incubated on ice for 1 hour with 200 μl of a 10 μg/ml solution of STRO-3 mAb in blocking buffer. The cells were then washed twice in HHF by centrifugation at 400 × g. A 1/50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer was added, and the cells were incubated on ice for 1 hour. Cells were washed twice in MACS buffer (Ca ²⁺ -free and Mn ²⁺ -free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) as above and resuspended in a final volume of 0.9 ml of MACS buffer.

100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice and resuspended in 0.5 ml of MACS buffer and then loaded onto a micro MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover unbound STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under the deposit number PTA-7282 - see International Publication No. WO2006/108229). After adding another 1 ml of MACS buffer, the column was removed from the magnet and TNAP ⁺ cells were isolated by positive pressure. Cell aliquots from each fraction were stained with streptavidin-FITC and assessed for purity by flow cytometry.

Example 3: Cells selected by STRO-3 mAb are STRO-1 ^bright cells

Experiments were designed to demonstrate the feasibility of using STRO-3 mAb as a single reagent for isolating cellular STRO-1 bright cells.

Given that the isotype of STRO-3 (IgG1) is different from that of STRO-1 (IgM), the ability of STRO-3 to identify clonal CFU-F was assessed by two-color FACS analysis based on co-expression with STRO-1 ⁺ cells isolated using the MACS program ( Figure 1 ). The dot matrix histogram represents 5×10 ⁴ events collected in the form of list mode data. The vertical and horizontal lines are set to a reactivity level of <1.0% of the mean fluorescence obtained with isotype-matched control antibodies 1B5 (IgG) and 1A6.12 (IgM) treated under the same conditions. The results indicate that a smaller population of STRO-1 ^bright cells co-expresses TNAP (upper right quadrant), while the remaining STRO-1 ⁺ cells fail to react with STRO-3 mAb. All four quadrants of cells isolated by FACS were subsequently analyzed for the incidence of CFU-F (Table 1).

Table 1: Enrichment of human bone marrow cells based on co-expression of the cell surface markers STRO-1 and TNAP by two-color FACS analysis (see Figure 1). FACS-sorted cells were cultured in αMEM supplemented with 20% FCS under standard cloning conditions. Data represent the mean number of colony-forming cells (CFU-F) on day 14 per 10 ^<5> cells plated ± SE (n=3 different bone marrow aspirates). These data demonstrate that human MPCs are restricted to the TNAP-positive portion of the BM, which clearly co-expresses the STRO-1 antigen.

Bone marrow Enrichment (increase fold) Ungraded BMMNC 11.0±2.2 1.0 4,511±185 410 0.0 0.0

Example 4: Relative gene and surface protein expression of STRO-1 ^dark and STRO-1 ^bright cells

In the first series of experiments, semi-quantitative RT-PCR analysis was used to examine the gene expression profiles of various lineage-associated genes expressed by STRO-1 ^Deep or STRO-1 ^Bright populations isolated by fluorescence-activated cell sorting ( FIG. 2A ). In the second series of experiments, flow cytometry and mean channel fluorescence analysis were used to examine the surface protein expression profiles of various lineage-associated proteins expressed by STRO-1 ^Deep or STRO-1 ^Bright populations isolated by fluorescence-activated cell sorting.

Total cellular RNA was prepared from 2×10 ⁶ STRO-1 ^bright or STRO-1 ^deep sorted primary cells, chondrocyte aggregates and other induced cultures and lysed using the RNAzolB extraction method (Biotecx Lab. Inc., Houston, TX) according to the manufacturer's recommendations. The RNA isolated from each subpopulation was then used as a template for cDNA synthesis using a first-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden). The expression of various transcripts was assessed by PCR amplification using a standard protocol as previously described (Gronthos et al., J. Bone and Min. Res. 14:48-57, 1999). The primer sets used in this study are shown in Table 2. After amplification, each reaction mixture was analyzed by 1.5% agarose gel electrophoresis and observed by ethidium bromide staining. RNA integrity was assessed by the expression of GAPDH.

ImageQant software was used to assess the relative gene expression of each cell marker with reference to the expression of housekeeping gene GAPDH (Fig. 2B, 2C). In addition, the protein expression profile of the MPC amplified in vitro was examined using dual-color flow cytometry analysis based on the expression of multiple cell lineage-related markers in combination with STRO-1 antibody. An overview of the general phenotype of the genes and protein expression of STRO-1 ^deep and STRO-1 ^bright cultured cells is shown in Table 3. The data show that the differential expression of the markers (including angiopoietin 1, VCAM-1, SDF-1, IL-1 _β , TNF α and RANKL) associated with perivascular cells of STRO-1 ^bright MPC amplified in vitro is higher. The comparison between the protein and gene expression profiles of STRO-1 ^deep and STRO-1 ^bright cultured cells is summarized in Tables 3 and 4.

Subtractive hybridization studies were also performed to identify genes uniquely expressed by STRO-1 ^bright cells. Briefly, STRO-1 ^dark and STRO-1 ^bright were separated as described above (see Figure 3 A). Total RNA was prepared from STRO-1 ^dark and STRO-1 ^bright cells collected from 5 different bone marrow samples using the RNA STAT-60 system (TEL-TEST). First chain synthesis was performed using the SMART cDNA synthesis kit (Clontech Laboratories). The mRNA/single-stranded cDNA hybrids were amplified according to the manufacturer's instructions using the specific primer sites on the 3' and 5' initial ends formed during the initial RT process by long-range PCR (Advantage 2 PCR kit; Clontech). After digesting the STRO-1 ^bright cDNA with RsaI, two aliquots were used to connect different specific linker oligonucleotides using the Clontech PCR-Select cDNA subtraction kit. Two rounds of subtractive hybridization were performed according to the manufacturer's protocol using STRO-1 ^bright (tester) and STRO-1 ^dark (driver) cDNA and vice versa. This procedure was also performed in reverse using the STRO-1 ^dark test cDNA hybridized to the STRO-1 ^light driver cDNA.

To identify genes uniquely expressed by the STRO-1 ^bright population, STRO-1 ^bright subtracted cDNA was used to construct replicate low-density microarray filters containing 200 randomly selected bacterial clones transformed with STRO-1 ^bright subtracted cDNA linked to a T/A cloning vector. The microarrays were then probed with [ ³² P] dCTP-labeled STRO-1 ^bright or STRO-1 ^deep subtracted cDNA ( FIG. 3B-3C ). Different screens identified a total of 44 clones that were highly differentially expressed between the STRO-1 ^deep and STRO-1 ^bright subpopulations. DNA sequencing of all differentially expressed clones revealed that only one clone represented a known stromal cell mitogen; i.e., platelet-derived growth factor (PDGF) (Gronthos and Simmons, Blood. 85:929-940, 1995). Interestingly, 6 of the 44 clones were found to contain DNA insertions corresponding to the chemokine stromal-derived factor 1 (SDF-1). The high abundance of SDF-1 transcripts in human STRO-1 ^bright cells was confirmed by semiquantitative RT-PCR of total RNA prepared from freshly sorted STRO-1 ^bright , STRO-1 ^dark , and STRO-1 ^negative bone marrow subsets (Fig. 3D and Table 3).

Table 2. RT-PCR primers and conditions for specific amplification of human mRNA

Table 3. Summary of relative gene expression in STRO-1 ^Bright and STRO-1 ^Deep populations. A list of genes showing measurable and differential expression between STRO-1 ^Bright and STRO-1 Deep populations when assayed by reverse transcription PCR is shown. Values represent relative gene expression with reference to the housekeeping gene GAPDH.

To correlate protein surface expression with the density of STRO-1 expression, single cell suspensions of cells obtained from bone marrow MPCs expanded in vitro were prepared by trypsin/EDTA separation and subsequently incubated with a combination of STRO-1 antibodies and antibodies that identify multiple cell lineage-associated markers. STRO-1 was identified using goat anti-mouse IgM-fluorescein isothiocyanate, while all other markers were identified using goat anti-mouse or anti-rabbit IgG-phycoerythrin. For those antibodies that identify intracellular antigens, cell preparations were first labeled with STRO-1 antibodies, fixed with 70% cold ethanol to permeabilize the cell membrane and then incubated with antibodies specific for the intracellular antigen. Isotype-matched control antibodies were used under the same conditions. Two-color flow cytometric analysis was performed using a COULTEREPICS flow cytometer and list mode data were collected. The dot plots represent 5,000 list mode events indicating the fluorescence intensity levels of each lineage cell marker (y-axis) and STRO-1 (x-axis). Vertical and horizontal quadrants were established with reference to isotype-matched negative control antibodies.

Table 4. Summary of relative protein expression in STRO-1 ^Bright and STRO-1 ^Dark populations. Shown is a list of proteins that showed differential expression between STRO-1 ^Bright and STRO-1 ^Dark populations when assayed by flow cytometry. Values represent the relative mean fluorescence intensity of the staining.

These results indicate that SDF-1α and RANKL are abundantly expressed by STRO-1 ^bright cells. This is important because these proteins are both known to be involved in the upregulation of CD4+CD25+ regulatory T cells, which confer protection against immune disorders such as GVHD (Loser et al., Nature Medicine 12:1372-1379, 2006; Hess, Biol. Blood Marrow Transplant, 12(1 Suppl 2):13-21, 2006; and Meiron et al., J. Exp. Medicine 205:2643-2655, 2008).

Example 5: In vitro immunosuppressive activity

To assess the immunosuppressive activity of culture-expanded STRO-1 ^bright cells (MPC (B)), we used CD3/CD28 stimulation as a readout. The results were compared to a population of culture-expanded bone marrow-derived STRO-1 negative cells (MSC (A)) isolated as in Example 1. Human peripheral blood mononuclear cells (PBMC) were stimulated with CD3/CD28-coated beads in the presence of four increasing concentrations of MSC and MPC preparations. T cell proliferation was measured by 3H-Tdr incorporation.

MSCs (A) and STRO- ¹ MPCs (B) were tested for their ability to inhibit the response of human peripheral blood mononuclear cells (PBMCs) to CD3/CD28 stimulation. MSCs and MPCs at varying ratios to PBMC cultures, or commercially available control human MSCs (Lonza), were added. After 3 days, 3H-Tdr was added for 18 hours and the cultures were then harvested.

The proliferation of PBMC in response to CD3/CD28 was inhibited in a dose-dependent manner by all preparations. However, preparation B was significantly superior to the effect produced by preparation A and control hMSC (Figure 4). At a 1:100 MSC:PBMC ratio, MPC B still inhibited 70% of control T cell proliferation, while control commercial MSC (Lonza) and MSC A produced 50% and 60% inhibition, respectively (Figure 5).

Example 6: Induction and Treatment of GvHD

The in vivo immunosuppressive activity of STRO-1 ^bright cells (MPC(B)) was studied using a graft-versus-host disease (GvHD) model based on multiple minor histocompatibility site mismatched donor-recipient pairs. Bone marrow mononuclear cells (BMMC) (5×10 ⁶ ) and spleen cells (30×10 ⁶ ) from B10.D2 (H2d) donors, from which T cells were removed, were injected intravenously into lethally irradiated (750 cGy) BALB/c (H2d) recipient mice. In this case, spleen lymphocytes from B10.D2 recognize and attack BALB/c recipient tissues and produce weight loss, fibrosis and hair loss. The disease was monitored using a conventional scoring system by weighing the animals and assessing skin manifestations 4-5 weeks after transplantation. As a comparison, the immunosuppressive activity of bone marrow-derived STRO-1 negative cells (MSC(A)) isolated as in Example 1 was evaluated.

While the positive control group of mice did not receive any further treatment, the experimental groups were intravenously injected with MSCs (A) or STRO- ¹ MPCs (B) at doses of 2×10 ⁶ , 1×10 ⁶ , or 0.3×10 ⁶ per mouse three times per week starting from week 4. The mice were monitored twice per week. Each group contained eight mice.

Example 7: Effects of MSCs and MPCs on the Development of GvHD

Mice received 1 or 2 x 10 ⁶ MSC A (A1 and A2), 1 or 2 x 10 ⁶ MPC B (B1 and B2). The kinetics of the disease in the absence or presence of MSC treatment are reported in Figure 6. After infusion of 1 x 10 ⁶ cells, there was a clear difference between the effects of B and A. Although mice receiving preparation A did not show any substantial differences from mice that did not receive cells, the group injected with preparation B showed a significant beneficial effect on disease severity. Thirteen weeks after transplantation, the average GvHD score of mice that had received B1 was 0.5, compared to 2.3 in the other groups.

We then investigated whether the anti-GvHD effect was dose-dependent. Therefore, according to the same model described for the previous doses, one group of mice was injected with a higher dose (2×10 ⁶ cells per mouse) and one group was injected with a lower dose (0.3×10 ⁶ cells per mouse) of A or B. Figure 7 reports the effect of the highest dose. The therapeutic effect of high-dose MPC (B2) was superior to that of A2, with no GvHD seen in this group during the first 11 weeks. At weeks 14 and 15, the consequences of GvHD per mouse were even more pronounced (Figure 8), with no mice surviving in the A1 group.

Finally, injection of a lower dose (0.3×10 ⁶ cells per mouse) again showed that STRO-1 ^bright MPCs (B) had a superior effect on reducing GVHD scores compared to A by week 9 ( Figures 9 and 10 ).

Data from this pilot study consistently demonstrated that STRO-1- ^bright MPCs exhibited enhanced immunosuppressive capacity compared to untreated or STRO-1-negative MSCs. This was evident in both in vitro and, more importantly, in vivo analyses. STRO-1- ^bright MPCs produced a significant clinical effect in preventing the indicated GvHD at doses ranging from 0.3 to 2 x ¹⁰ cells per mouse.

The present invention relates to the following embodiments:

1. A method for preventing the development of GvHD complications or treating said GvHD complications in a mammalian patient, said method comprising administering to said mammal a cell population enriched in STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from said STRO-1 ^bright cells and/or their progeny.

2. A method as described in embodiment 1 above, wherein the method comprises administering to the mammal (a) precursors of myeloid lineage cells, and (b) a cell population enriched in STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny.

3. The method of embodiment 2, wherein the cell population enriched in STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells are administered to the mammal prior to administering the precursors of the myeloid lineage cells.

4. The method of embodiment 2, wherein the cell population enriched in STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells are co-administered with the precursors of the myeloid lineage cells.

5. The method of any one of embodiments 2 to 4, wherein the precursors of the myeloid lineage cells are allogeneic cells administered to the mammal to treat a malignant or genetic disease of the blood.

6. The method of any one of embodiments 1 to 5, wherein the STRO-1 ^bright cells and/or their progeny are allogeneic.

7. The method of any one of embodiments 1 to 6, wherein the cell population enriched in STRO-1 ^bright cells and/or their progeny cells and/or the soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells are administered systemically.

8. The method of embodiment 7, wherein the cell population enriched for STRO-1 ^bright cells and/or progeny cells thereof and/or the soluble factors obtained from the STRO-1 ^bright cells and/or progeny cells thereof are administered by intravenous injection.

9. The method of any one of embodiments 1 to 8, comprising administering between 0.1×10 ⁶ and 5×10 ⁶ STRO-1 ^bright cells and/or progeny thereof.

10. The method of any one of embodiments 1 to 9, comprising administering between 0.3×10 ⁶ and 2×10 ⁶ STRO-1 ^bright cells and/or progeny thereof.

11. The method of any one of embodiments 1 to 10, comprising administering a low dose of STRO-1 ^bright cells and/or progeny thereof.

12. The method of embodiment 11, wherein the low dose of STRO-1 ^bright cells and/or progeny thereof comprises between 0.1×10 ⁵ and 0.5×10 ⁶ STRO-1 ^bright cells and/or progeny thereof.

13. The method of embodiment 11, wherein the low dose of STRO-1 ^bright cells and/or progeny thereof comprises about 0.3×10 ⁶ STRO-1 ^bright cells and/or progeny thereof.

14. The method of any one of embodiments 1 to 13, wherein the population enriched for STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny are administered once a week or less frequently.

15. The method of any one of embodiments 1 to 14, wherein the mammal is suffering from aplastic anemia, myelofibrosis, or bone marrow failure following chemotherapy and radiation therapy.

16. The method of any one of embodiments 1 to 15, further comprising administering an immunosuppressive drug to the mammal.

All references cited in this document are incorporated herein by reference.

It will be appreciated by those skilled in the art that various changes and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit and scope of the broad description of the present invention. Therefore, the embodiments of the present invention are considered to be illustrative and not restrictive in all aspects.

Claims (15)

1. Use of a cell population enriched with STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from said STRO-1 ^bright cells and/or their progeny in the preparation of a medicament for the prevention of the development of GvHD complications in mammalian patients or for the treatment of said GvHD complications. 2. The use as described in claim 1, wherein the drug is suitable for administering (a) a precursor of bone marrow lineage cells, and (b) a cell population enriched with STRO-1 ^bright cells and/or their progeny, and/or a soluble factor derived from said STRO-1 ^bright cells and/or their progeny, to said mammal. 3. The use as claimed in claim 2, wherein the drug is adapted to administer to the mammal, prior to administration of the precursor of the bone marrow lineage cells, the cell population enriched with STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells. 4. The use as claimed in claim 2, wherein the drug is suitable for co-administration of the cell population enriched with STRO-1 ^bright cells and/or their progeny cells and/or a soluble factor obtained from the STRO-1 ^bright cells and/or their progeny cells with a precursor of the bone marrow lineage cells. 5. The use as described in any one of claims 2 to 4, wherein the precursor of the bone marrow lineage cells is an allogeneic cell for the treatment of malignant or hereditary diseases of the blood. 6. The use as described in any one of claims 1 to 4, wherein the STRO-1 ^bright cells and/or their progeny are allogeneic. 7. The use according to any one of claims 1 to 4, wherein the drug is suitable for systemic administration of the cell population enriched with STRO-1 ^bright cells and/or their progeny cells and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny cells. 8. The use as claimed in claim 7, wherein the drug is adapted to administer, by intravenous injection, the cell population enriched with STRO-1 ^bright cells and/or their progeny cells and/or soluble factors derived from the STRO-1 ^bright cells and/or their progeny cells. 9. The use as claimed in any one of claims 1 to 4, wherein the drug is suitable for administration at a dose between 0.1 × ^10⁶ and 5 × ^10⁶ STRO-1 ^bright cells and/or their progeny. 10. The use as claimed in any one of claims 1 to 4, wherein the drug is suitable for administration at a dose between 0.3 × ^10⁶ and 2 × ^10⁶ STRO-1 ^bright cells and/or their progeny. 11. The use as described in any one of claims 1-4, wherein the drug is suitable for administration at a dose between 0.1 × ^10⁵ and 0.5 × ^10⁶ STRO-1 ^bright cells and/or their progeny. 12. The use as claimed in claim 11, wherein the drug is suitable for administration at a dose of about 0.3 × ^10⁶ STRO-1 ^bright cells and/or their progeny. 13. The use as claimed in any one of claims 1 to 4, wherein the drug is adapted to administer, once weekly or at a lower frequency, the population enriched with STRO-1 ^bright cells and/or their progeny and/or soluble factors obtained from the STRO-1 ^bright cells and/or their progeny. 14. The use as claimed in any one of claims 1 to 4, wherein the mammal is suffering from aplastic anemia, myelofibrosis, or bone marrow failure following chemotherapy and radiation therapy. 15. The use as described in any one of claims 1 to 4, wherein the cell population enriched with STRO-1 ^bright cells and/or their progeny and/or the soluble factors obtained from said STRO-1 ^bright cells and/or their progeny are administered in combination with an immunosuppressive drug.