HK1214166B - Immunomodulatory properties of multipotent adult progenitor cells and uses thereof - Google Patents

Description

Immunomodulatory properties of multipotent adult progenitor cells and their uses

This application is a divisional application of the PCT application with an international application date of November 9, 2006, an international application number of PCT/US2006/043804, an application number entering the national phase of 200680050862.9, and an invention name of “Immune regulatory properties of multipotent adult progenitor cells and their uses”.

Citation of Related Applications

This application is a continuation-in-part of U.S. application serial number 11/269,736, filed November 9, 2005; which is a continuation-in-part of U.S. application serial number 11/151,689, filed June 13, 2005; which is a continuation-in-part of U.S. application serial number 10/963,444, filed October 11, 2004 (abandoned); which is a continuation-in-part of U.S. application serial number 10/048,757, filed February 1, 2002; which is a continuation-in-part of U.S. application serial number 10/048,757, filed August 4, 2000, and filed February 15, 2001 in English in WO U.S. national phase application PCT/US00/21387, published on 01/11011; PCT/US00/21387 claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 60/147,324, filed on August 5, 1999, and U.S. Provisional Application Serial No. 60/164,650, filed on November 10, 1999, and is a continuation-in-part of U.S. Application Serial No. 10/467,963, filed on August 11, 2003; U.S. national phase application PCT/US00/21387, published on 01/11011, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 60/147,324, filed on August 5, 1999, and to U.S. Provisional Application Serial No. 60/164,650, filed on November 10, 1999, and is a continuation-in-part of U.S. Application Serial No. 10/467,963, filed on February 14, 2002, and filed in English on August 22, 2002 in WO No. 60/343,836 filed on October 25, 2001, all of which are hereby incorporated by reference in their entireties and from which this application claims the full benefit of priority.

Technical Field

The field of the invention is immunomodulation of multipotent adult progenitor cells ("MAPCs") and the use of these cells to modulate immune responses in primary and adjunctive therapies.

Background Art

The therapeutic use of organ transplants, including bone marrow transplants, has steadily increased since its inception and has become an important treatment option for a wide range of diseases, including but not limited to hematological disorders, immune disorders, and malignant diseases.

Unfortunately, the therapeutic benefits of transplantation are often complicated, rendered ineffective, or prevented by adverse immune responses to the transplant. The most prominent adverse reactions encountered with transplantation therapy are (i) host-versus-graft ("HVG") (rejection of the transplant by an immunocompetent host) and (ii) graft-versus-host disease ("GVHD") (a process that primarily occurs in an immunocompromised host when the host is recognized as non-self by the immunocompetent cells of the transplant).

Of course, transplant rejection in the host can be avoided by perfectly matching the donor and the host. However, with the exception of autologous tissue, only identical twins can be expected to be truly isogenic. A perfect match between a donor from one individual and a host/recipient from another does not actually exist. Therefore, the use of autologous tissue is the only other way to achieve a perfect match. Unfortunately, host tissue is often not suitable or is not isolated before it is needed. The need for transplant therapy is often actually to replace damaged tissue in the host. Therefore, the use of isogenic tissue, while an effective way to solve the problem of adverse host response to transplanted tissue, is generally not useful in practical applications.

If syngeneic matching is not possible, the adverse immune effects of transplantation therapy can be mitigated by matching the allogeneic donor and host as closely as possible. Blood and/or tissue typing is used to match the donor and host to provide the highest probability of treatment success. However, even the closest matching of allogeneic tissues does not prevent the development of severe HVG, so transplantation therapy involves the use of immunosuppression and immunosuppressive drugs, which are discussed below.

Another approach that has been implemented to avoid HVG complications in transplantation therapy is to disable the recipient's immune system. This has been achieved through the use of radiation therapy and/or immunosuppressive chemotherapy and/or antibody therapy. The resulting suppression of the host's immune response is often very effective in helping the transplant (e.g., bone marrow) establish in the host. However, immunoablation or suppression can compromise the host's immune defenses. This can make the host highly susceptible to infection after even the slightest exposure to an infectious agent. The resulting infection is a major cause of morbidity and mortality in transplant patients.

Compromising the host immune system can also create or exacerbate another serious problem encountered in transplantation therapy: graft-versus-host disease ("GVHD"). GVHD occurs when donor tissue contains immunocompetent cells that recognize the recipient's MHC proteins as non-self. This activates T cells, causing them to secrete cytokines such as IL-2 (interleukin-2), IFN (interferon gamma), and TNFα (tumor necrosis factor alpha). These signals trigger an immune attack against recipient targets, including the skin, gastrointestinal tract, liver, and lymphoid organs (Ferrara and Deeg, 1991). GVHD is particularly problematic in bone marrow transplantation, where it has been shown to be primarily mediated by T lymphocytes (Grebe and Streilein, 1976). In fact, approximately 50% of bone marrow transplant patients develop acute GVHD. Many of these patients die (15%-45%).

There are also other immune system dysfunctions, disorders, and diseases that occur as primary lesions or as secondary effects of other lesions and/or their treatment. These include tumors, bone marrow lesions, blood lesions, autoimmune diseases, and some inflammatory diseases, which are discussed further below. Primary and adjunctive therapies for these disorders and diseases, such as those for HVG and GVHD, often involve the use of immunosuppressive drugs. All current therapies have drawbacks and side effects.

Immunosuppressive drugs

Considerable effort has been devoted to developing drugs that treat these immune system dysfunctions and thereby ameliorate or eliminate their deleterious effects without causing additional deleterious side effects. Some progress has been made toward this goal, with a variety of drugs being developed and used to prevent and/or treat these dysfunctions. The introduction of these more effective drugs marks a tremendous advance in the medical practice of transplantation therapy; however, none of these drugs is ideal. Indeed, none of the immunosuppressive drugs currently available for clinical use in transplantation therapy is completely effective. All of the drugs have serious drawbacks and adverse side effects, which are summarized below. For a review, see Farag (2004), "Chronic graft-versus-host disease: where do we go from here?" Bone Marrow Transplantation 33:569-577.

Corticosteroids, primarily used to treat inflammation and inflammatory diseases, are known to have immunosuppressive properties and are considered by many to be the best primary treatment for HVG and GVHD. They inhibit T cell proliferation and T cell-dependent immune responses, at least in part, by suppressing the expression of certain cytokine genes involved in T cell activation and T cell-dependent immune responses.

Cyclosporine is one of the most commonly used drugs for immunosuppression and prevention of HVG and GVHD. It is generally a potent immunosuppressant. While it effectively reduces adverse immune responses in transplant patients, it also significantly weakens the immune system, making patients highly susceptible to infection. Consequently, patients are more susceptible to pathogen exposure and less able to mount an effective immune response. Consequently, even mild pathogens can be life-threatening. Cyclosporine can also cause a variety of other undesirable side effects.

Methotrexate, alone or in combination with other drugs, is also widely used for the prevention and treatment of HVG and GVHD. Studies have shown that, even if it is effective, it is clearly no more effective than cyclosporine. Like cyclosporine, methotrexate can cause a variety of side effects, some of which can be harmful to patients.

FK-506 is a macrolide-like compound. Similar to cyclosporine, it is also derived from fungal sources. Cyclosporine and FK-506 have similar immunosuppressive effects. They block the early events of T cell activation by forming heterodimeric complexes with their respective cytoplasmic receptor proteins (i.e., cyclophilin and FK-binding protein). This then inhibits the phosphatase activity of calcineurin, ultimately inhibiting the expression of nuclear regulatory proteins and T cell activation genes.

Other drugs that have been used for immunosuppression include antithymocyte globulin, azathioprine, and cyclophosphamide. These have proven to be unhelpful. Rapamycin, another macrolide-like compound that interferes with T cell responses to IL-2, has also been used to block T cell-activated immune responses. RS-61443, a mycophenolic acid derivative, has been shown to inhibit allograft rejection in experimental animals. Mizoribine, an imidazole nucleoside, blocks the purine biosynthesis pathway and inhibits mitogen-stimulated T and B cell proliferation in a manner similar to azathioprine and RS-61443. Deoxyspergualin, a synthetic analog of spergualin, has been shown to exert immunosuppressive properties in preclinical transplant models. The antimetabolite brequinar sodium is an inhibitor of dihydroorotate dehydrogenase, blocking the formation of both uridine and cytidine nucleosides by inhibiting pyrimidine synthesis. Berberine and its pharmacologically tolerable salts have been used as immunosuppressants to treat autoimmune diseases such as rheumatism, to treat allergies, and to prevent transplant rejection. Berberine has been reported to inhibit B cell antibody production and generally suppress humoral immune responses without affecting T cell proliferation. See Japanese Patent No. 07-316051 and U.S. Patent No. 6,245,781.

No matter these immunosuppressive drugs are used alone or in combination with other agents, none of them is completely effective. All of them usually make patients still prone to HVG and GVHD, weakening the ability of patients to resist infection. This makes them more susceptible to infection and more unable to fight infection when infection occurs. In addition, all of these drugs can cause serious side effects, including for example gastrointestinal toxicity, nephrotoxicity, hypertension, bone marrow suppression, liver toxicity, hypertension and gingival hypertrophy etc. None of them proves to be fully acceptable or effective therapeutic drugs. In short, in view of these shortcomings, for adverse immune system dysfunction and/or response such as HVG and GVHD, there is currently no fully satisfactory treatment based on drugs.

It has long been thought that more specific types of immunosuppression could be developed that do not have these drawbacks. For example, agents that specifically suppress or eliminate alloreactive T cells would be effective against HVG and GVHD (at least for allogeneic transplants) without the adverse side effects of agents that systemically attack and compromise the immune system. However, such agents have yet to be developed.

Use of restricted stem cells in transplantation

The use of stem cells as an alternative to or in conjunction with immunosuppressants has recently attracted attention. There have been some encouraging observations in this area. In recent years, a variety of stem cells have been isolated and characterized. These range from stem cells with highly restricted differentiation potential and limited ability to grow in culture to stem cells with apparently unrestricted differentiation potential and unlimited ability to grow in culture. The former are generally easier to derive and can be obtained from a variety of adult tissues. The latter must be derived from germ cells and embryos and are referred to as embryonic stem ("ES") cells, embryonic germ ("EG") cells, and germ cells. Embryonic stem ("ES") cells have unlimited self-renewal capacity and can differentiate into all tissue types. ES cells are derived from the inner cell mass of the blastocyst. Embryonic germ ("EG") cells are derived from primordial germ cells of the post-implantation embryo. Stem cells derived from adult tissues are of limited value because they are immunogenic, have limited differentiation potential, and have a limited ability to reproduce in culture. ES, EG, and germ cells do not have these disadvantages, but they have a pronounced tendency to form teratomas in xenogeneic hosts, which has raised significant concerns about their use in medical therapy. Therefore, while their broad differentiation potential is advantageous, there is pessimism about their usefulness in clinical applications. Embryonic stem cells are also subject to ethical controversies that may hinder their use in treating disease.

Some efforts to find alternatives to ES, EG, and germ cells have focused on cells derived from adult tissues. Although adult stem cells have been identified in most mammalian tissues, their differentiation potential is restricted, much narrower than that of ES, EG, and germ cells. Indeed, many of these cells can only give rise to one or a few differentiated cell types, and many others are restricted to a single embryonic lineage.

For example, hematopoietic stem cells can only differentiate into cells of the hematopoietic lineage, neural stem cells differentiate only into cells of neuroectodermal origin, and mesenchymal stem cells ("MSCs") are limited to cells of mesenchymal origin. Due to the limitations, risks, and controversies associated with ES, EG, and germ cells mentioned above, a large portion of the work involving the use of stem cells in transplantation therapy has utilized MSCs. Results from recent years appear to indicate that allografts of MSCs do not produce the HVG immune response that is always seen when other tissues are transplanted between allogeneic individuals. Furthermore, results suggest that MSCs can, at least in some cases, attenuate the lymphocyte immune response.

While these results directly suggest that MSCs may be useful in reducing HVG and/or GVHD, which often accompany allogeneic transplantation, the observed immunosuppressive effects of MSCs were highly dose-dependent, requiring relatively high doses to be observed. Indeed, in an in vitro mixed lymphocyte assay, the reduction in lymphocyte proliferation was only “significant” when the MSCs:lymphocyte ratio was 1:10 or greater. Furthermore, the observed suppressive effect decreased and became unobservable as the dose of MSCs was decreased, and the presence of MSCs actually appeared to stimulate T cell proliferation at ratios below 1:100. The same dose effect was also observed in a mitogen-stimulated lymphocyte proliferation assay. For reviews, see Ryan et al. (2005) "Mesenchymal stem cells avoid allogeneic rejection," J. Inflammation 2:8; Le Blanc (2003) "Immunomodulatory effects of fetal and adult mesenchymal stem cells," Cytotherapy 5(6):485-489 and Jorgensen et al. (2003) "Engineering mesenchymal stem cells for immunotherapy," Gene Therapy 10:928-931. Additional results are summarized below.

For example, Bartholomew and colleagues found that baboon MSCs did not stimulate the proliferation of allogeneic lymphocytes in vitro and that MSCs reduced the proliferation of mitogen-stimulated lymphocytes by more than 50% in an in vitro mixed lymphocyte assay. They further demonstrated that in vivo administration of MSCs prolonged skin graft survival (relative to controls). Both the in vitro and in vivo results required high doses of MSCs: a 1:1 ratio of MSCs to lymphocytes was required for the in vitro results. Practically speaking, the amount of MSCs required to approximate this ratio in humans may be prohibitively high. This could limit the application of MSCs. See Bartholomew et al. (2002): "Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo," Experimental Hematology 30: 42-48.

Maitra and colleagues investigated the effect of human MSCs on the engraftment of allogeneic human umbilical cord blood cells after co-infusion into sublethally irradiated NOD-SCID mice. They found that human MSCs promoted engraftment in an in vitro proliferation assay without stimulating allogeneic T cells. They also found that human MSCs inhibited in vitro activation of allogeneic human T cells by mitogens. These effects were dose-dependent, requiring relatively high ratios for inhibition (Maitra et al. (2004) Bone Marrow Transplantation 33:597-604).

Recently, Le Blanc and his colleagues reported that a patient with usually fatal grade IV acute GVHD was successfully treated by giving "third party haploidentical" MSCs. The patient was a 9-year-old boy with acute lymphoblastic leukemia in third remission. Initially, the patient was treated with radiation and cyclophosphamide, and then given blood cells that were exactly the same as his own cells at the HLA-A, HLA-B and HLA-DR beta loci. These cells were obtained from an unrelated female donor. Despite active treatment, including the administration of multiple immunosuppressants, the patient developed grade IV acute GVHD 70 days after transplantation. He often suffered from invasive bacterial, viral and fungal infections.

Under these apparently dire circumstances, an alternative blood stem cell transplant was attempted. Haploidentical MSCs were isolated from the patient's mother and expanded in vitro for three weeks. The cells were harvested and administered intravenously to the patient at 2 x 10 ⁶ cells/kg. There were no signs of toxicity related to the MSCs and no major side effects. Many symptoms subsided within a few days after the transplant, but residual disease remained evident. After several more intravenous injections of MSCs using the same method, the patient's symptoms and GVHD completely subsided. The patient remains disease-free one year after being free of the disease. In the authors' experience, this patient is unusual in surviving such a severe case of GVHD. The results reported by Le Blanc et al. are both promising and encouraging and should serve as an incentive for the development of effective therapies using stem cells. Le Blanc et al. (2004) "Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymalstem cells," Lancet 363: 1439-41.

Still, these results, including those by Le Blanc and colleagues, reveal potential drawbacks to MSCs. The cells need to be administered alongside traditional immunosuppressive modalities, which can continue to generate harmful immune responses. MSCs also require very high doses to be effective, which can lead to higher costs, more difficult administration, and a greater risk of toxicity and other side effects, among other drawbacks.

Given these limitations of current stem cell-based transplantation-related therapies, there is clearly a strong need for progenitor cells that can be used in all, or at least most, of the recipient hosts without requiring host-recipient haplotype matching. Furthermore, there is a need for cells that have a greater "specific activity," making them therapeutically effective at lower doses and that can be administered without the problems associated with the high-dose regimens required to achieve beneficial results with MSCs. There is also a need for cells that have virtually unlimited differentiation potential, giving rise to cells that are present in the organism of interest.

Therefore, there is a continuing need for cells that possess the self-renewal and differentiation capabilities of ES, EG, and germ cells but are not immunogenic; do not form teratomas when transplanted homologously or xenogeneically into a host; do not pose other safety issues associated with ES, EG, and germ cells; retain the other advantages of ES, EG, and germ cells; are easily isolated from readily available sources such as placenta, umbilical cord, umbilical cord blood, blood, and bone marrow; can be safely stored for long periods of time; are readily available without risk to volunteers, donors, or patients and without the need for consent from others; and do not present the technical and marketing difficulties associated with their acquisition and use with ES, EG, and germ cells.

Recently, a class of cells referred to herein as multipotent adult progenitor cells ("MAPCs") have been isolated and characterized (see, e.g., U.S. Patent No. 7,015,037, which is incorporated herein by reference in its entirety). ("MAPCs" may also be referred to as "MASCs"). These cells offer many of the advantages of ES, EG, and germ cells without many of their disadvantages. For example, MAPCs can be cultured indefinitely without losing their differentiation potential. They demonstrate efficient, long-term engraftment and differentiation along multiple developmental lineages of NOD-SCID mice without signs of teratoma formation (common in ES, EG, and germ cells) during engraftment and differentiation (Reyes, M. and CM Verfaillie (2001) Ann NY Acad Sci .938:231-5).

Summary of the Invention

Thus, in some embodiments of the present invention, the present invention provides cells that (i) are not embryonic stem cells, not embryonic germ cells, and not germ cells; (ii) are capable of differentiating into at least one cell type of each of at least two of the endoderm, ectoderm, and mesoderm embryonic lineages; (iii) do not induce a harmful immune response when introduced into a non-syngeneic subject; and (iv) are capable of modulating an immune response when introduced into a subject. In certain embodiments of this aspect, the present invention provides cells that, in addition to being as described above, also have immunosuppressive properties. Furthermore, various embodiments of the present invention provide cells that are the aforementioned cells having immunomodulatory properties that can be used to treat, such as to eliminate, prevent, ameliorate, alleviate, reduce, minimize, eliminate, and/or regulate harmful immune responses and/or processes in a host. In some embodiments of the present invention, the cells are used alone or in combination with other therapeutic agents and modalities for this purpose as a primary treatment modality. In some embodiments of the present invention, the cells are used in an adjuvant therapy modality, in which they are used as the sole therapeutic agent or in combination with other therapeutic agents. In some embodiments of the invention, the cells are used in one or more primary treatment modalities and in one or more adjunctive treatment modalities, either alone or in combination with other therapeutic agents or modalities.

The cells of the present invention will be described in more detail herein and are generally referred to herein as "multipotent adult progenitor cells," abbreviated as "MAPC" (singular) and "MAPCs" (plural). It should be understood that these cells are not ES cells, EG cells, or germ cells, and have the ability to differentiate into cell types of at least two of the three primitive germ layer lineages (ectoderm, mesoderm, and endoderm), for example, into cells of all three primitive lineages.

For example, MAPCs can form the following cells and other cells of their lineages: splanchnic mesoderm cells, muscle cells, bone cells, chondrocytes, endocrine cells, exocrine cells, endothelial cells, germinal cells, odontogenic cells, visceral mesoderm cells, hematopoietic cells, stromal cells, bone marrow stromal cells, neurons, neuroectoderm cells, epithelial cells, ocular cells, pancreatic cells and hepatocyte-like cells, among others. The cells formed by MAPCs include osteoblasts, chondroblasts, adipocytes, skeletal muscle cells, skeletal myocytes, biliary epithelial cells, pancreatic acinar cells, mesangial cells, smooth muscle cells, cardiac muscle cells, cardiomyocytes, osteocytes, angiogenic cells, oligodendrocytes, neurons (including serotonergic, GABAergic, and dopaminergic neurons), glial cells, microglia, pancreatic epithelial cells, intestinal epithelial cells, liver epithelial cells, skin epithelial cells, renal epithelial cells, pancreatic islet cells, fibroblasts, hepatocytes, and other cells of the same lineage as the above cells, etc.

MAPCs possess telomerase activity, which is essential for self-renewal and is thought to be required for maintaining an undifferentiated state. They also typically express oct-3/4. Oct-3/4 (oct-3A in humans) is also unique to ES, EG, and germ cells. It is considered a marker of undifferentiated cells with extensive differentiation potential. Oct-3/4 is also generally believed to play a role in maintaining cells in an undifferentiated state. Oct-4 (oct-3 in humans) is a transcription factor found in pre-gastrulation embryos, early cleavage embryos, cells of the inner cell mass of the blastocyst, and embryonal carcinoma (EC) cells (Nichols, J. et al. (1998) Cell 95:379-91). It is downregulated when cells are induced to differentiate. The oct-4 gene (oct-3 in humans) is transcribed into at least two splice variants in humans, oct-3A and oct-3B. The oct-3B splice variant is found in many differentiated cells, while the oct-3A splice variant (previously also referred to as oct-3/4) is reported to be specific for undifferentiated embryonic stem cells. See Shimozaki et al. (2003) Development 130:2505-12. The expression of oct-3/4 plays an important role in determining the early steps in embryogenesis and differentiation. Together with rox-1, oct-3/4 leads to transcriptional activation of the zinc finger protein rex-1, which is also required for maintaining ES cells in an undifferentiated state (Rosfjord, E. and Rizzino, A. (1997) Biochem Biophys Res Commun 203:1795-802; Ben-Shushan, E. et al. (1998) Mol Cell Biol 18:1866-78).

MAPCs often express other markers thought to be unique to primitive stem cells. These include rex-1, rox-1, and sox-2. Rex-1 is controlled by oct-3/4, which activates the downstream expression of rex-1. Rox-1 and sox-2 are expressed in non-ES cells.

Various embodiments of the present invention provide methods of using MAPCs to eliminate, prevent, treat, improve, alleviate, reduce, minimize, eliminate and/or remediate diseases and/or adverse immune responses and/or processes in a host. Certain embodiments of the present invention provide methods of using the cells themselves as a primary therapeutic modality. In some embodiments of the present invention, the cells are used as a primary therapeutic modality together with one or more other agents and/or therapeutic modalities. In some embodiments of the present invention, the cells are used as an adjuvant therapeutic modality, that is, as an adjuvant modality to another primary therapeutic modality. In some embodiments, the cells are used as the sole active agent in an adjuvant therapeutic modality. In other embodiments, the cells are used as an adjuvant therapeutic modality together with one or more other agents or therapeutic modalities. In some embodiments, the cells are used as both primary and adjuvant therapeutic agents and/or modalities. In both aspects, the cells can be used alone in the primary modality and/or in the adjuvant modality. They can also be used together with other therapeutic agents or modalities in the primary modality, in the adjuvant modality, or in both modalities.

As described above, a primary treatment, such as a therapeutic agent, therapy, and/or treatment modality, is a primary abnormality, such as a disease, that is targeted (i.e., intended to act on). Adjunctive therapies, such as therapies, and/or treatment modalities, can be administered together with a primary treatment, such as a therapeutic agent, therapy, and/or treatment modality, to act on the primary abnormality, such as the disease, and to supplement the effect of the primary treatment, thereby increasing the overall efficacy of the regimen. Adjunctive therapies, such as therapeutic agents, therapies, and/or treatment modalities, can also be administered to act on complications and/or side effects of the primary abnormality, such as the disease, and/or complications and/or side effects caused by a treatment, such as a therapeutic agent, therapy, and/or treatment modality. For any of these applications, one, two, three, or more primary treatments can be used together with one, two, three, or more adjunctive therapies.

In some embodiments, MAPCs are administered to a subject before a dysfunction, such as a disease, side effects, and/or adverse immune response, develops. In some embodiments, the cells are administered while the dysfunction is developing. In some embodiments, the cells are administered after the dysfunction has been established. The cells can be administered at any stage during the development, persistence, and/or spread of the dysfunction, or after the dysfunction has resolved.

As previously mentioned, embodiments of the present invention provide cells and methods for primary or adjuvant therapy. In certain embodiments of the present invention, the cells are given to allogenic subjects. In some embodiments, they are autologous to the subject. In some embodiments, they are syngeneic to the subject. In some embodiments, the cells are xenogeneic to the subject. Whether allogenic, autologous, syngeneic or xenogeneic, in various embodiments of the present invention, MAPCs have weak immunogenicity or are non-immunogenic in the subject. In some embodiments, MAPCs have sufficiently low immunogenicity or are non-immunogenic, and fully do not have general harmful immune responses, so that when given to allogenic subjects, they can be used as "universal" donor cells without the need for tissue typing and matching. According to various embodiments of the present invention, MAPCs also can be stored and maintained in a cell bank, so that they can be available for use when needed.

In all of these aspects and other aspects, embodiments of the present invention provide MAPCs from mammals, which in one embodiment are humans, and in other embodiments are non-human primates, rats and mice, and dogs, pigs, goats, sheep, horses, and cattle. MAPCs prepared from the above mammals can be used in all of the methods and other aspects of the present invention described herein.

MAPCs according to various embodiments of the present invention can be isolated from multiple compartments and tissues of such mammals, including but not limited to bone marrow, blood, spleen, liver, muscle, brain, and other compartments and tissues mentioned below. In some embodiments, MAPCs are cultured before use.

In some embodiments, MAPCs are genetically engineered, for example to improve their immunomodulatory properties. In some embodiments, genetically engineered MAPCs are produced by in vitro culture. In some embodiments, genetically engineered MAPCs are produced from transgenic organisms.

In various embodiments, MAPCs are administered to a subject by any route that effectively delivers a cell therapy agent. In some embodiments, the cells are administered by injection, including local injection and/or systemic injection. In certain embodiments, the cells are administered in the middle of and/or near the site of the malfunction they are intended to treat. In some embodiments, the cells are administered by injection at a location that is not near the malfunction site. In some embodiments, the cells are administered by systemic injection, such as intravenous injection.

In some embodiments, MAPCs are administered once, twice, three times, or more than three times, until the desired therapeutic effect is achieved or their administration no longer appears to provide benefit to the subject. In some embodiments, MAPCs are administered continuously for a period of time, such as by intravenous infusion. MAPCs can be administered for a short period of time, for a period of days, for a period of weeks, for a period of months, for a period of years, or for a longer period of time.

The following numbered paragraphs describe exemplary embodiments of the present invention that illustrate certain aspects and features of the present invention. They are not intended to be exhaustive of the many aspects and embodiments of the present invention and, therefore, are not intended to limit the present invention in any way. Many other aspects, features, and embodiments of the present invention are described herein. Those skilled in the art will readily recognize many other aspects and embodiments upon reading this application and considering it appropriately in light of the prior art and knowledge in the art.

The following numbered paragraphs are self-referential. The phrase "according to any preceding or following" refers to all preceding and all following numbered paragraphs and their contents. All phrases of the form "according to #" are direct references to that numbered paragraph, e.g., "according to 46" refers to paragraph 46 of the set of numbered paragraphs herein. All cross-references are combinatorial except where redundant or inconsistent in scope. Cross-references are expressly used to provide a concise description indicating that various combinations of the subject matter are included.

1. A method for treating immune dysfunction in a subject, the method comprising:

To a subject who may be susceptible to, is susceptible to, or has been susceptible to an immune dysfunction, cells (MAPCs) are administered via an effective route and in an amount effective to treat the immune dysfunction that: are not embryonic stem cells, embryonic germ cells, or germ cells; are capable of differentiating into at least one cell type of each of at least two of the endoderm, ectoderm, and mesoderm embryonic lineages; do not induce a harmful immune response in the subject; and are effective to treat the immune dysfunction.

2. A method for adjuvant treatment of a subject, the method comprising administering to a subject who may suffer from, is suffering from, or has suffered from an immune dysfunction, via an effective route and in an amount effective to treat the immune dysfunction, cells (MAPCs) that are not embryonic stem cells, embryonic germ cells, or germ cells; are capable of differentiating into at least one cell type of each of at least two of the endoderm, ectoderm, and mesoderm embryonic lineages; do not cause a harmful immune response in the subject; and are effective to treat the immune dysfunction, wherein the cells are administered in adjuvant to one or more other therapies administered to the subject to treat the same thing, to treat something else, or to treat both.

3. A method according to any preceding or following description, wherein the cell is capable of differentiating into at least one cell type of each of the endoderm, ectoderm and mesoderm embryonic lineages.

4. A method according to any preceding or following method, wherein the cell is capable of expressing telomerase.

5. A method according to any preceding or following description, wherein the cells are positive for oct-3/4.

6. The method according to any preceding or following description, wherein the cells undergo at least 10-40 cell doublings in culture prior to administration to the subject.

7. A method according to any preceding or following description, wherein the cell is a mammalian cell.

8. A method according to any preceding or following description, wherein the cell is a human, horse, cow, goat, sheep, pig, rat or mouse cell.

9. A method according to any preceding or following description, wherein the cell is a human, rat or mouse cell.

10. A method according to any preceding or following description, wherein the cell is a human cell.

11. A method according to any preceding or following, wherein the cells are derived from cells isolated from any of the following: placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymus tissue, spinal cord tissue, adipose tissue and liver tissue.

12. The method according to any preceding or following description, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow, blood and spleen tissue.

13. A method according to any preceding or following description, wherein the cells are derived from cells isolated from any one of placental tissue, umbilical cord tissue, umbilical cord blood, bone marrow or blood.

14. A method according to any preceding or following description, wherein the cells are derived from any one or more isolated cells from bone marrow or blood.

15. A method according to any preceding or following description, wherein the cells are allogeneic to the subject.

16. A method according to any preceding or following description, wherein the cells are xenogeneic to the subject.

17. A method according to any preceding or following description, wherein the cells are autologous to the subject.

18. A method according to any preceding or following description, wherein the subject is a mammal.

19. The method according to any preceding or following description, wherein the subject is a mammalian companion animal, a mammalian livestock animal, a mammalian research animal, or a non-human primate.

20. A method according to any preceding or following description, wherein the subject is a human.

21. The method according to any preceding or following description, wherein said cells are administered to the subject in one or more doses ^{comprising 104-108} said cells per kilogram of subject mass.

22. The method according to any preceding or following description, wherein said cells are administered to the subject in one or more doses ^{comprising 105-107} said cells per kilogram of subject mass.

23. The method according to any preceding or following description, wherein said cells are administered to the subject in one or more doses comprising 5 x ¹⁰⁶ - 5 x ¹⁰⁷ said cells per kilogram of subject mass.

24. The method according to any preceding or following description, wherein said cells are administered to the subject in one or more doses comprising 2 x ¹⁰⁷ to 4 x ¹⁰⁷ said cells per kilogram of subject mass.

25. A method according to any preceding or following description, wherein in addition to the cells, one or more factors are administered to the subject.

26. A method according to any preceding or following description, wherein in addition to the cells, one or more growth factors, differentiation factors, signal transduction factors and/or factors that increase homing are administered to the subject.

27. A method according to any preceding or following description, wherein in addition to the cells, one or more cytokines are administered to the subject.

28. The method according to any preceding or following description, wherein the cells are administered to the subject adjunctively with another therapy administered before, simultaneously with, or after administration of the cells.

29. The method according to any preceding or following description, wherein the cells are administered to the subject in conjunction with administration of one or more immunosuppressive agents to the subject.

30. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a transplant, and wherein said cells are administered in adjunct to said transplant.

31. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a kidney, heart, lung, liver or other organ transplant, wherein said cells are administered in adjunct to said transplant.

32. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a transplant of bone marrow, vein, artery, muscle or other tissue, wherein said cells are administered in adjunct to said transplant.

33. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a transplant of blood cells, pancreatic islet cells or other tissue or organ regenerative cells, wherein said cells are administered in adjunct to said transplant.

34. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a blood cell transplant, wherein said cells are administered in adjunct to said transplant.

35. A method according to any preceding or following description, wherein in addition to treatment with said cells, the subject is to receive or has received a bone marrow transplant, wherein said cells are administered in adjunct to said transplant.

36. A method according to any preceding or following description, wherein the subject has been, will be or is being treated with one or more immunosuppressants in addition to treatment with said cells, wherein said cells are administered adjunctively to treatment with said immunosuppressants.

37. A method according to any preceding or following, wherein the subject has been treated, will be treated or is being treated with one or more of the following in addition to treatment with the cells: corticosteroids, cyclosporine A, cyclosporine-like immunosuppressants, cyclophosphamide, anti-thymocyte globulin, azathioprine, rapamycin, FK-506 and macrolide-like immunosuppressants other than FK-506, rapamycin and immunosuppressive monoclonal antibody agents (i.e., such immunosuppressive agents are immunosuppressive monoclonal antibodies or agents comprising all or one or more portions of monoclonal antibodies, such as chimeric proteins comprising the Fc or Ag binding sites of monoclonal antibodies), wherein the cells are administered in adjunct to one or more of the above treatments.

38. The method of any preceding or following, wherein the subject has been treated, will be treated, or is being treated with one or more of the following in addition to treatment with the cells: corticosteroids, cyclosporine A, azathioprine, rapamycin, cyclophosphamide, FK-506, or an immunosuppressive monoclonal antibody agent, wherein the cells are administered adjunctively to one or more of the above treatments.

39. The method according to any preceding or following description, wherein the cells are administered to the subject in conjunction with administration of one or more antibiotic agents to the subject.

40. The method according to any preceding or following description, wherein the cells are administered to the subject in conjunction with administration of one or more antifungal agents to the subject.

41. The method according to any preceding or following description, wherein the cells are administered to the subject in conjunction with administration of one or more antiviral agents to the subject.

42. A method according to any preceding or following, wherein the cells are administered to the subject in conjunction with administration of any combination of two or more of any immunosuppressive agent and/or antibiotic agent and/or antifungal agent and/or antiviral agent to the subject.

43. A method according to any preceding or following description, wherein the cells are administered to a subject as an adjunct to transplantation therapy to treat a host-versus-graft reaction in the subject that is impairing or may impair the therapeutic efficacy of the transplant and/or is causing or may cause transplant rejection.

44. A method according to any preceding or following description, wherein the cells are administered to a subject having one or more of a weakened immune system, such as a deficient immune system and/or an ablated immune system.

45. A method according to any preceding or following description, wherein the cells are administered to the subject in adjunct to radiotherapy or chemotherapy, or a combination of radiotherapy and chemotherapy, which has been administered, is being administered or will be administered to the subject.

46. A method according to any preceding or following description, wherein the cells are administered to the subject adjunctively to an ongoing regimen of radiotherapy or chemotherapy or a combination of radiotherapy and chemotherapy.

47. A method according to any preceding or following description, wherein the subject's immune system has been weakened, compromised and/or ablated by radiotherapy, chemotherapy or a combination of radiotherapy and chemotherapy.

48. A method according to any preceding or following description, wherein the subject is a recipient of a non-syngeneic blood cell or bone marrow transplant, the subject's immune system has been weakened or ablated by radiation therapy, chemotherapy, or a combination of radiation therapy and chemotherapy, and the subject is at risk of developing or has developed graft-versus-host disease.

49. A method according to any preceding or following description, wherein the subject is a recipient of a non-syngeneic blood cell or bone marrow transplant, the subject's immune system has been weakened or ablated by radiation therapy, chemotherapy, or a combination of radiation therapy and chemotherapy, and immunosuppressive drugs administered to the subject, wherein in addition the subject is at risk of developing graft-versus-host disease or has already developed the disease, and the cells are administered to the subject adjunctively to one or more other therapies (i.e., transplantation, radiation therapy, chemotherapy and/or immunosuppressive drugs) to treat the graft-versus-host disease.

50. A method according to any preceding or following description, wherein the subject is to be or is currently the recipient of a non-syngeneic transplant, is at risk of developing a host versus graft reaction or has developed such a reaction, and wherein the cells are administered to treat the host versus graft reaction.

51. The method according to any preceding or following description, wherein the subject is at risk of or currently suffering from a tumor and the cells are administered to assist in the treatment of the tumor.

52. The method according to any preceding or following description, wherein the subject is at risk of or suffering from a tumor of blood or bone marrow cells and the cells are administered to assist in the treatment of the tumor.

53. The method according to any preceding or following description, wherein the subject is at risk of or currently suffering from a benign tumor of bone marrow cells, a myeloproliferative disorder, a myelodysplastic syndrome, or an acute leukemia and the cells are administered as an adjunct to the treatment of such a disorder.

54. The method according to any preceding or following description, wherein the subject is at risk of or suffering from a benign tumor of bone marrow cells and the cells are administered to assist in the treatment of the tumor.

55. The method according to any preceding or following description, wherein the subject is at risk of or suffering from a myeloproliferative disorder and the cells are administered adjunctively to the treatment of the disorder.

56. The method of any preceding or following, wherein the subject is at risk of or currently suffering from one or more of the following diseases: chronic myeloid leukemia ("CML") (also known as chronic granulocytic leukemia ("CGL"), agnogenic myelofibrosis, essential thrombocythemia, polycythemia vera, or other myeloproliferative disorders, and the cells are administered as an adjunct to the treatment of such diseases.

57. The method according to any preceding or following description, wherein the subject is at risk of or suffering from a myelodysplastic syndrome and the cells are administered adjunctively to the treatment of the syndrome.

58. A method according to any preceding or following description, wherein the subject is at risk of or suffering from acute leukemia and the cells are administered adjunctively to the treatment of the disease.

59. A method according to any preceding or following, wherein the subject is at risk of or currently suffering from one or more of the following diseases: acute multiple myeloma, myeloblastic leukemia, chronic myelogenous leukemia ("CML"), acute promyelocytic leukemia, pre-B acute lymphoblastic leukemia, chronic lymphocytic leukemia ("CLL"), B cell lymphoma, hairy cell leukemia, myeloma, T acute lymphoblastic leukemia, peripheral T cell lymphoma, other lymphoid leukemia, other lymphoma or other acute leukemia, and the cells are administered as an adjunct to the treatment of these diseases.

60. A method according to any preceding or following description, wherein the subject is at risk of or suffering from anemia or other blood disorder and the cells are administered as an adjunct to the treatment of the disorder.

61. The method according to any preceding or following description, wherein the subject is at risk of or currently suffering from a hemoglobinopathy, thalassemia, bone marrow failure syndrome, sickle cell anemia, aplastic anemia, Fanconi anemia, or immune hemolytic anemia, and the cells are administered as an adjunct to the treatment of such disease.

62. A method according to any preceding or following, wherein the subject is at risk of or suffering from one or more of the following diseases: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, chronic myelomonocytic leukemia or other myelodysplastic syndromes, and the cells are administered as an adjunct to the treatment of these diseases.

63. The method according to any preceding or following description, wherein the subject is at risk of or suffering from Fanconi anemia and the cells are administered adjunctively to the treatment of the disease.

64. The method according to any preceding or following description, wherein the subject is at risk of or suffering from an immune dysfunction and the cells are administered to assist in the treatment of the immune dysfunction.

65. The method according to any preceding or following description, wherein the subject is at risk of or suffering from an innate immune deficiency and the cells are administered in adjunct to the treatment of the innate immune deficiency.

66. A method according to any preceding or following description, wherein the subject is at risk of or currently suffering from an autoimmune abnormality, disorder or disease, and the cells are administered in adjunct to the treatment of the abnormality, disorder or disease.

67. The method of any preceding or following, wherein the subject is at risk of or currently suffering from one or more of the following autoimmune disorders: Crohn's disease, Guillain-Barré syndrome, lupus erythematosus (also known as "SLE" and systemic lupus erythematosus), multiple sclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, diabetes mellitus (type I), Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome ("APS"), opsoclonus-myoclonus syndrome ("OMS"), temporal arteritis, acute disseminated encephalomyelitis ("ADEM" and "ADE"), Goodpasture's syndrome, Wegener's granulomatosis, celiac disease, pemphigus, polyarthritis, and warm autoimmune hemolytic anemia, and the cells are administered adjunctively to the treatment of the autoimmune disorders.

68. The method of any preceding or following, wherein the subject is at risk of or currently suffering from one or more of the following autoimmune disorders: Crohn's disease, lupus erythematosus (also known as "SLE" and systemic lupus erythematosus), multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease, diabetes mellitus (type I), Reiter's syndrome, primary biliary cirrhosis, celiac disease, polyarthritis, and warm autoimmune hemolytic anemia, and the cells are administered as an adjunct to the treatment of these autoimmune disorders.

69. The method of any preceding or following, wherein the subject is at risk of or currently suffering from one or more of the following diseases believed to have an autoimmune component: endometriosis, interstitial cystitis, neuromyotonia, scleroderma, progressive systemic scleroderma, vitiligo, vulvodynia, Chagas' disease, sarcoidosis, chronic fatigue syndrome, and dysautonomia, and the cells are administered adjunctively to the treatment of these diseases.

70. The method according to any preceding or following description, wherein the subject is at risk of or suffering from an inflammatory disease and the cells are administered in adjunct to the treatment of the inflammatory disease.

71. A method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more other pharmaceutically active agents.

72. A method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more other immunosuppressive agents.

73. A method according to any preceding or following, wherein the cells are administered in a formulation comprising one or more of the following agents: corticosteroids, cyclosporine A, cyclosporine-like immunosuppressants, cyclophosphamide, anti-thymocyte globulin, azathioprine, rapamycin, FK-506 and macrolide-like immunosuppressants other than FK-506, rapamycin, and immunosuppressive monoclonal antibody agents.

74. The method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more of the following agents: corticosteroids, cyclosporine A, azathioprine, cyclophosphamide, rapamycin, FK-506, and an immunosuppressive monoclonal antibody agent.

75. A method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more antibiotic agents.

76. A method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more antifungal agents.

77. A method according to any preceding or following description, wherein the cells are administered in a formulation comprising one or more antiviral agents.

78. A method according to any preceding or following description, wherein the cells are administered to the subject parenterally.

79. A method according to any preceding or following, wherein the cells are administered to the subject by any one or more of the following parenteral routes: intravenous, intraarterial, intracardiac, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intradermal, intradermal, subcutaneous and intramuscular injection.

80. The method according to any preceding or following description, wherein the cells are administered to the subject by any one or more of the following parenteral routes: intravenous, intraarterial, intradermal, intradermal, subcutaneous and intramuscular injection.

81. A method according to any preceding or following description, wherein the cells are administered to the subject by any one or more of the following parenteral routes: intravenous, intraarterial, intradermal, subcutaneous and intramuscular injection.

82. A method according to any preceding or following description, wherein the cells are administered to the subject by syringe via a hypodermic needle.

83. A method according to any preceding or following description, wherein the cells are administered to the subject via a catheter.

84. A method according to any preceding or following description, wherein the cells are administered to the subject by surgical implantation.

85. The method according to any preceding or following description, wherein the cells are administered to the subject by implantation using an arthroscopic procedure.

86. A method according to any preceding or following description, wherein the cells are administered to a subject in or on a support.

87. A method according to any preceding or following description, wherein the cells are administered to the subject in encapsulated form.

88. A method according to any preceding or following, wherein the cells are suitably formulated for administration by any one or more of the following routes: oral, rectal, epidermal, ocular, intranasal and pulmonary.

89. A method according to any preceding or following description, wherein the cells are administered to the subject in one dose.

90. A method according to any preceding or following description, wherein the cells are administered sequentially to the subject in a series of two or more doses.

91. A method according to any preceding or following description, wherein the cells are administered in a single dose, in two doses, or in more than two doses, wherein each dose is the same or different and is administered with equal or unequal intervals between them.

92. A method according to any preceding or following description, wherein the cells are administered over a period of less than one day to one week, one week to one month, one month to one year, one year to two years, or more than two years.

93. A method for treating an immune dysfunction in a subject, the method comprising administering to a subject suffering from an immune dysfunction, by a route and in an amount effective to treat the immune dysfunction in the subject, cells that: are not embryonic stem cells, embryonic germ cells, or germ cells; are capable of differentiating into at least one cell type of each of at least two of the endoderm, ectoderm, and mesoderm embryonic lineages; do not elicit a deleterious immune response in the subject; and are effective to treat the immune dysfunction in the subject.

94. A method of adjunctive therapy for treating an immune dysfunction in a subject, the method comprising administering to a subject suffering from an immune dysfunction, by a route and in an amount effective to treat the subject's immune dysfunction, cells that are not embryonic stem cells, embryonic germ cells, or germ cells; are capable of differentiating into at least one cell type of each of at least two of the endoderm, ectoderm, and mesoderm embryonic lineages; do not elicit a deleterious immune response in the subject; and are effective to treat the subject's immune dysfunction, wherein the cells are administered to the subject in adjunctive with one or more other therapies being administered to the subject to treat the same immune dysfunction, to treat one or more other dysfunctions, or both.

Other aspects of the invention are described in or are obvious from the following disclosure, and these other aspects are within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphic representation of a transcriptional profiling study performed to generate (identify) gene- and surface receptor-based markers that distinguish MAPCs of the present invention from other more lineage committed stem and progenitor cells. The experiment generated a panel of 75 markers that had a 10-fold differential expression between MSC cultures and MAPCs.

Fig. 2 is a group of graphs showing the three-lineage differentiation of rat MAPCs marked with GFP. The results show that MAPCs can differentiate into cells of all three embryonic lineages. As further described below, for endothelial differentiation, MAPCs were cultured on a fibronectin-coated plate in the presence of vascular endothelial growth factor B (VEGF-B). For hepatocyte differentiation, cells were grown on a matrigel-coated plate and treated with fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF). Neuronal differentiation was induced by sequentially treating with basic FGF (bFGF), FGF-8, Sonic Hedgehog (SHH), and brain-derived neurotrophic factor (BDNF). After two weeks, mRNA was extracted from the cells and applied to qPCR analysis using primers that can specifically detect various lineage markers. In all assays, cells cultured in the absence of cytokines that can induce lineage served as controls. The expression levels of each lineage marker were first normalized to the expression level of the internal control gene (GAPDH) that was not affected during differentiation. Differentiation success was then assessed by calculating the relative expression of differentiated or control cells compared to the levels of the parental rat cell line, with a cutoff level of more than a 5-fold increase in relative expression as a successful differentiation. Differentiated rat MAPCs showed significant expression of the endothelial markers von Willebrand factor and PECAM-1 (upper panels); the hepatic markers albumin, cytokeratin-18, and HNF-1a (middle panels); and the neuronal/astrocyte markers GFAP, stemness protein, and NF-200 (lower panels).

Figure 3 is a pair of bar graphs showing the low immunogenicity (upper panel) and immunosuppressive properties (lower panel) of MAPCs in a mixed lymphocyte reaction (MLR) described further below. In the upper panel: B+B = donor B + donor B; B+A = donor B + donor A; B+K = donor B + donor K; B+R = donor B + donor R; B+T = donor B + donor T; donor B + PHA; B+BMPC = donor B + MAPC. The same results were obtained for twelve different donors. In the lower panel: donor W + donor W; donor W + donor A; donor W + donor T; donor W + MSC; donor W + MAPC (17); donor W + PHA; donor W + donor A + MSC; donor W + donor A + MAPC (17); donor W + donor T + MSC; donor W + donor T + MAPC (17); donor W + donor P + MSC; donor W + donor P + MAPC (17). PHA is a phytohemagglutinin (positive control for T cell activation).

Figure 4 is a graph showing that MAPCs can inhibit the proliferation of ConA-stimulated T cells as described in Example 6. The inset text "LN only" represents the results of a control reaction without MAPCs. The number after MAPCs indicates how many cells were used in the assay.

FIG5A is a graph showing the immunosuppressive effect of Lewis MAPCs in a mixed lymphocyte reaction as described in Example 7. The inset in FIG5A lists the number of MAPCs in each reaction. In the inset, R represents responder cells and S represents stimulator cells (irradiated splenocytes from DA rats).

Figure 5B is a graph showing the immunosuppressive effect of Sprague-Dawley MAPCs in a mixed lymphocyte reaction as described in Example 7. Illustration text and abbreviations are the same as for Figure 5A.

FIG6 is a graph showing that the infusion of MAPCs did not adversely affect the health of the recipients as measured by their respiratory rate, and is further described in Example 8.

FIG7 is a bar graph depicting the results of an experiment demonstrating the ability of MAPCs to suppress an ongoing immune response. The graph shows that MAPCs are strongly immunosuppressive in MLRs whether added simultaneously with a T cell activator (stimulator) (day 0, left side of the graph) or added 3 days after the T cell activator (stimulator) (day 3, right side of the graph). The details of the experiment are further described in Example 10.

Figure 8 is a bar graph showing that MAPCs reversibly inhibit T cell proliferation in MLRs. Results are presented as mean cpm +/- SD of triplicate cultures. This graph is further described in Example 11.

FIG9 is a graph showing that MAPCs can suppress GVHD as described in Example 13.

definition

Certain terms used herein have the meanings set forth below.

A singular noun refers to one or more of the thing it refers to; at least one.

"Assisting in" means jointly, together, additionally, jointly, etc.

"Co-administration" can include simultaneous or sequential administration of two or more agents.

"Cytokine" refers to a cellular factor that can induce or enhance cell motility, such as homing of MAPCs or other stem cells, progenitor cells, or differentiated cells. Cytokines can also stimulate such cells to divide.

As used herein, "harmful" means capable of causing damage. For example, as used herein, a "harmful immune response" refers to an immune response that can cause damage, such as a deficient or too weak immune response, an overly strong immune response, and/or a misdirected immune response. Harmful immune responses include immune responses that occur in immune diseases and can cause damage. Examples include a lack of an immune response in immunodeficiency diseases, an excessive and/or misdirected immune response in autoimmune diseases. Harmful immune responses also include immune responses that interfere with medical treatment, including immune responses that are otherwise normal and that interfere with medical treatment. Examples include immune responses involved in rejection of a transplant, and responses of immunocompetent cells in a transplant that can cause graft-versus-host disease.

"Differentiation factor" refers to a factor, such as a growth factor, that induces lineage commitment of a cell.

As used herein, "dysfunction" refers to a disorder, disease, or deleterious effect of an otherwise normal process. For example, immune dysfunction includes immune diseases such as autoimmune diseases and immunodeficiency. It also includes immune responses that interfere with medical therapy, including otherwise normal immune responses that interfere with medical therapy. Examples of such dysfunction include immune responses that contribute to transplant rejection and responses of immunocompetent cells in the transplant that can cause graft-versus-host disease.

"EC cells" refers to embryonal carcinoma cells.

"Effective amount" generally refers to an amount that can provide the desired local or systemic effect. For example, an effective amount is an amount sufficient to achieve a favorable or desired clinical result. The effective amount can be provided all at once in a single administration, or divided into several doses to provide the effective amount. For example, an effective amount of MAPCs can be given in one or more administrations and can include any pre-selected cell amount. To accurately determine how much amount is considered an effective amount, it depends on factors specific to each subject, including their weight, age, loss and/or disease or injury being treated, and the length of time since the injury or disease occurred. Those skilled in the art will be able to determine the effective amount for a given subject based on these conventional considerations in the art. Thus, for example, those skilled in the art, such as physicians, based on the known properties of MAPCs disclosed herein and in the art, while taking into account the above factors, will be able to determine the effective amount of MAPCs for a given subject. "Effective dose" as used herein is synonymous with "effective amount."

"EG cells" refers to embryonic germ cells.

"Engraftment" refers to the process by which cells come into contact with existing tissues of interest in the body and become incorporated therein.

"Enriched colonies" refers to a relative increase in the number of MAPCs relative to other cells or components in the starting colony, such as an increase in the number of MAPCs relative to one or more non-MAPC cell types in culture (eg, primary culture) or in vivo.

"ES cells" refer to embryonic stem cells.

"Expansion" refers to the multiplication of a cell or cells without differentiation.

As used herein, "Fanconi anemia" is synonymous with Fanconi anemia, a hereditary disease.

"GVHD" refers to graft-versus-host disease, a process that occurs primarily in an immunocompromised host when the host is recognized as non-self by the immunocompetent cells of the transplant.

"HVG" refers to host versus graft reaction, which refers to the process that occurs when a host rejects a transplant. Typically, HVG is initiated when a transplant is recognized as foreign (non-self) by the host's immune-competent cells.

"Isolated" refers to a cell or cells that is no longer associated with one or more cells or one or more cellular components with which it is associated in vivo.

"MAPC" is the acronym for "multipotent adult progenitor cells". It refers to non-ES, non-EG, non-germ cells that can produce more than one germ layer, such as cell lineages of all three germ layers (i.e., endoderm, mesoderm, and ectoderm). MAPCs also have telomerase activity. They can be oct-3/4 (e.g., human oct-3A) positive. They can also express rex-1 and rox-1. In addition, they can express sox-2, SSEA-4 and/or nanog. The term "adult" in MAPC is not restrictive. It simply means that these cells are not ES, EG, or germ cells. Generally, MAPC used herein is singular and MAPCs is plural. MAPCs are also called multipotent adult stem cells (MASCs). See U.S. Patent 7,015,037, the disclosure of which regarding MAPC/MASC and its isolation and growth methods is incorporated herein by reference.

“MASC” See MAPC.

"MNC" refers to mononuclear cells.

"Mode" refers to a type, approach, avenue, or method, such as a treatment mode, which is a type of therapy.

"MSC" is the acronym for mesenchymal stem cells.

"Multipotency" in MAPCs refers to the ability to give rise to cell lineages of more than one germ layer upon differentiation, such as all three primitive germ layers (ie, endoderm, mesoderm, and ectoderm).

"Persistence" refers to the ability of cells to resist rejection and maintain and/or increase in number in vivo over time (eg, days, weeks, months, or years).

"Progenitor cells" as used in the context of multipotent adult progenitor cells (MAPCs) means that these cells are capable of giving rise to other cells, such as further differentiated cells. The term is non-restrictive and does not limit these cells to a particular lineage.

"Self-renewal" refers to the ability to generate replicon stem cells with the same differentiation potential as the stem cell from which the replicon stem cell was generated. A similar term used in this context is "proliferation."

A "subject" is a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, domesticated animals, competitive animals, and pets. Subjects in need of treatment using the methods of the present invention include those suffering from a disorder, malfunction, or disease (e.g., immunodeficiency or malfunction, such as HVG and GVHD), or side effects of these problems, or treatments for these problems, which may benefit from the administration of MAPCs as a primary or adjunctive therapy.

As used herein, "graft" refers to a cell, tissue, or organ that is introduced into a subject. The graft can be derived from the subject, derived from culture, or derived from a source other than the subject.

"Treatment" includes treating, preventing, ameliorating, inhibiting or remediating a defect, malfunction, disease or other process that would cause adverse effects, such as an immune system defect, malfunction, disease or other process that would adversely affect the function or properties of the immune system or that would interfere with therapy.

DETAILED DESCRIPTION

introduction

MAPCs, whether used alone or in combination with other therapeutics, are very promising for treating diseases by cell transplantation techniques, such as for tissue and organ regeneration. Potential obstacles to achieving the promise of MAPCs for treating diseases and for tissue and organ regeneration are adverse immune responses that typically complicate or hinder the success of transplant therapies such as blood and bone marrow transplantation and solid organ transplantation. Notable among these immune complications are transplant rejection by the host immune system (referred to herein as host-versus-graft reaction, "HVG") and systemic damage to the immunocompromised host (referred to herein as graft-versus-host disease, "GVHD") when the immunocompetent cells in the transplant are activated due to contact with non-self components of the host.

It has been found (described in more detail elsewhere herein) that MAPCs do not elicit an immune response in allogeneic hosts. Therefore, transplantation of MAPCs into allogeneic hosts should not result in allogeneic graft rejection (ie, HVG).

Furthermore, we found that allogeneic MAPCs could be administered to the host at high concentrations without adverse effects on respiration, suggesting that there was no inappropriate accumulation and/or deposition in the lungs.

It has also been discovered (described in greater detail elsewhere herein) that MAPCs can modulate immune responses. Specifically in this regard, MAPCs have been found to suppress immune responses, including but not limited to those involving, for example, HVG and GVHD (to name two examples). More specifically in this regard, MAPCs have been found to suppress T cell proliferation even in the presence of potent T cell stimulators such as concanavalin A and allogeneic stimulator cells.

Furthermore, it was found that even relatively small numbers of MAPCs could suppress these responses. Indeed, as little as 3% MAPCs were sufficient to reduce T cell responses to potent stimulators in vitro by 50% in a mixed lymphocyte reaction.

Thus, certain aspects of the invention in this regard provide, inter alia, compositions and methods for treating, ameliorating and/or regulating or eliminating adverse immune responses, such as those that occur in transplantation therapy.

The low immunogenicity of allogeneic MAPCs, their ability to suppress adverse immune responses, and their high specific activity make them particularly valuable as adjuvant therapies for the treatment of diseases with adverse immune components. Such diseases include autoimmune diseases, which are usually caused by dysfunction of the subject's own immune system. MAPCs can also be used as immunosuppressive adjuvant therapy agents to treat adverse immune responses that occur in transplantation therapies. Examples include HVG in immunocompetent hosts and GVHD in immunodeficient hosts. MAPCs can also be used as adjuvant immunosuppressive therapies in the treatment of various tumors, anemias, and blood diseases, and in the treatment of certain inflammatory diseases. Diseases in this regard will be discussed in detail below.

Using the methods of MAPC isolation, characterization, and expansion described herein, combined with the disclosure herein regarding the immunosuppressive properties of MAPCs, MAPCs can be used to prevent, inhibit, or reduce immune disorders, malfunctions, or diseases, including, for example, adverse immune responses, such as those caused by other therapies, including adverse immune responses that complicate transplantation therapy, such as HVG and GVHD. Such disorders, malfunctions, and diseases also include innate immune diseases and autoimmune diseases, among others.

MAPCs can be used as primary and adjunctive therapeutic agents and modalities in these and other aspects. MAPCs can be used therapeutically alone or in combination with other agents. MAPCs can be given before, during, and/or after the administration of such other agents. Similarly, whether used alone or in combination with other agents, MAPCs can be given before, during, and/or after the transplant is performed. If given during the transplant process, MAPCs can be given together with the transplant material, or separately. If given separately, MAPCs can be given sequentially or simultaneously with the transplant. In addition, MAPCs can be given before and/or after the transplant.

Other agents that can be used in conjunction with MAPCs, particularly in transplantation therapy, include immunomodulators. A variety of such agents are described elsewhere herein. In certain embodiments of the present invention, the immunomodulator is an immunosuppressant, such as those described elsewhere herein. Such agents include corticosteroids, cyclosporine A, cyclosporine-like immunosuppressive compounds, azathioprine, cyclophosphamide, and methotrexate.

MAPCs can be administered to a host by a variety of methods discussed elsewhere herein. In certain embodiments, MAPCs are administered by injection, such as by intravenous injection. In some embodiments, MAPCs are encapsulated for administration. In some embodiments, MAPCs are administered in situ. Examples include in situ administration of MAPCs in solid organ transplantation and in organ repair. These and other forms of administration are discussed below.

In some embodiments of the present invention, MAPCs are administered at a dose measured by the ratio of MAPCs (cells) to body mass (weight). Alternatively, MAPCs can be administered at a dose of a fixed number of cells. Dosages, routes of administration, formulations, etc. are discussed in more detail elsewhere herein.

Mechanism of action

Although not wanting to be limited to any one or more explanatory mechanisms about the immunomodulatory properties and other properties, activities and effects of MAPCs, it is worth mentioning that they can regulate immune responses through a variety of modes. For example, MAPCs can have a direct effect on the graft or host. This direct effect is mainly a matter of direct contact between the cells of the MAPCs and the host or graft. This contact can be contact with the structural members of the cell or contact with components in the cell's adjacent environment. This direct mechanism can involve direct contact, diffusion, absorption (uptake) or other processes well known to those skilled in the art. The direct activity and effect of MAPCs may be spatially limited to, for example, local deposition areas or limited to the body compartments reached by injection.

MAPCs can also "home" in response to "homing" signals, such as those released at sites of injury or disease. Because homing is often mediated by signals whose natural function is to recruit cells to sites in need of repair, homing behavior can be a powerful tool for focusing MAPCs on therapeutic targets. This effect can be stimulated by specific factors, as discussed below.

MAPCs can also regulate immune processes through their response to various factors. This can occur in addition or alternatively to direct regulation. Such factors may include homing factors, mitogens, and other stimulatory factors. They may also include differentiation factors and factors that can trigger specific cellular processes. The latter include factors that can prompt cells to secrete other specific factors, such as factors involved in recruiting cells such as stem cells (including MAPCs) to the site of injury or disease.

In addition or alternatively to the above situation, MAPCs can be secreted to act on endogenous cells such as stem cells or progenitor cells. These factors can act on other cells to cause, increase, reduce or inhibit their activity. MAPCs can secrete factors that can act on stem cells, progenitor cells or differentiated cells to promote these cells to divide and/or differentiate. MAPCs that home to the site in need of repair can secrete nutritional factors that can attract other cells to the site. In this way, MAPCs can attract stem cells, progenitor cells or differentiated cells to the site in need of repair. MAPCs can also secrete factors that can promote these cells to divide or differentiate.

The secretion of such factors, including trophic factors, can contribute to the efficacy of MAPCs in, for example, limiting inflammatory damage, limiting vascular permeability, improving cell survival, and inducing and/or increasing the homing of repair cells to the site of damage. Such factors can also directly affect T cell proliferation. Such factors can also affect dendritic cells by reducing their phagocytic and antigen presentation activities, which can also affect T cell activity.

Through these and other mechanisms, MAPCs can provide favorable immunomodulatory effects, including but not limited to the inhibition of unwanted and/or adverse immune reactions, responses, functions, diseases, etc. The MAPCs in various embodiments of the present invention can provide favorable immunomodulatory properties and effects, which can be used by themselves or in combination with adjuvant therapy to eliminate, prevent, alleviate, reduce, improve, alleviate, treat, eliminate and/or regulate adverse immune processes and/or conditions. Such processes and conditions include, for example, autoimmune diseases, anemia, tumors, HVG, GVHD and certain inflammatory diseases, which are described in more detail elsewhere herein. MAPCs can be used for these other aspects, especially in mammals. In various embodiments of the present invention in this regard, MAPCs are often used therapeutically in human patients in conjunction with other therapies.

Administration of MAPC

MAPC products

MAPCs can be prepared from a variety of tissues, such as bone marrow cells, as described in more detail elsewhere herein.

In many embodiments, MAPC preparations are clonally derived. In principle, the MAPCs in these preparations are genetically identical to each other and, if prepared and maintained properly, are free of other cells.

In some embodiments, MAPC preparations that are less pure than these can be used. Colonies of lower purity may occur when the initial cloning step requires more than one cell, but this is rare. If these cells are not all MAPCs, then expansion will produce mixed colonies, in which MAPCs are only one of at least two types of cells. More often, mixed colonies are produced when MAPCs are mixed with one or more other types of cells.

In many embodiments, the purity of MAPCs administered to a subject is about 100%. In other embodiments, it is 95%-100%. In some embodiments, it is 85%-95%. Particularly in the case of mixing with other cells, the percentage of MAPCs can be 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%.

The quantity of MAPCs in a given volume can be measured by known and conventional procedures and instruments. The percentage of MAPCs in a cell mixture of a given volume can be measured by almost the same procedure. Cells can be easily counted by hand or using an automatic cell counter. Using specific staining and naked eye inspection, and by using the automatic method of a specific binding reagent (normally an antibody, fluorescent marker) and a fluorescence-activated cell sorter, the specific cells in a given volume can be measured.

MAPC immunomodulation can involve undifferentiated MAPCs. It can involve MAPCs that are committed to a certain differentiation pathway. Such immunomodulation can also involve MAPCs that have differentiated into less potent stem cells with limited differentiation potential. It can also involve MAPCs that have differentiated into terminally differentiated cell types. The optimal type and mixture of MAPCs will be determined by their specific use case, and it will be a routine design issue for those skilled in the art to determine the effective type or combination of MAPCs.

preparation

The selection of a formulation for administering MAPCs for a given application depends on a variety of factors. Chief among these factors are the species of the subject, the nature of the disorder, dysfunction, or disease being treated and its status and distribution in the subject, the nature of other therapies and agents being administered, the optimal route of administration of the MAPCs, the survival rate of the MAPCs when administered via that route, the dosage regimen, and other factors apparent to those skilled in the art. Specifically, for example, the selection of a suitable carrier and other additives depends on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is to be formulated as a solution, suspension, gel, or other liquid dosage form, such as a time-release dosage form or a liquid-filled form).

For example, cell survival may be an important determinant of the efficacy of cell-based therapies. This is true for both primary and adjuvant therapies. If the target site is not suitable for cell implantation and cell growth, this is another problem. This can hinder therapeutic MAPCs from approaching the site and/or moving into the site. Various embodiments of the present invention include measures to increase cell survival and/or overcome the problems caused by cell implantation and/or growth disorders.

Examples of compositions comprising MAPCs include liquid products, and liquid products include suspensions and products for intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may comprise a mixture of MAPCs with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, and the like. The composition may also be freeze-dried. The composition may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, gelling agents or viscosity enhancing additives, preservatives, flavorings, coloring agents, and the like, depending on the route of administration and the desired product. Suitable products may be prepared with reference to authoritative textbooks such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985 (incorporated herein by reference) without having to do too much experimentation.

The compositions of the present invention are often conveniently provided as liquid products, such as isotonic aqueous solutions, suspensions, emulsions, or viscous compositions, which can be buffered to a selected pH. Liquid products are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, the administration of liquid compositions is somewhat more convenient, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to extend the contact time with a particular tissue.

Often, various additives are included to improve the stability, sterility, and isotonicity of the composition, such as antimicrobial preservatives, antioxidants, chelating agents, and buffers. Protection against the effects of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, isotonic agents such as sugars and sodium chloride are preferably included. Prolonged absorption of the injectable form can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the present invention, any medium, diluent, or additive used should be compatible with cells.

MAPC solutions, suspensions, and gels typically contain, in addition to cells, a large amount of water (preferably purified sterile water). Small amounts of other ingredients may also be present, such as pH adjusters (e.g., bases such as NaOH), emulsifiers or dispersants, buffers, preservatives, wetting agents, and gelling agents (e.g., methylcellulose).

Typically, compositions will be isotonic, meaning that they will have the same osmotic pressure as blood and tear fluid when properly prepared for administration.

The desired isotonicity of the compositions of the present invention can be achieved with sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred, especially for buffers containing sodium ions.

If necessary, the viscosity of the composition can be maintained at a selected level by an acceptable thickening agent of available medicine. Preferred methylcellulose is because it is easy to obtain, economical and easy to operate. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer etc. The preferred concentration of the thickening agent will depend on the selected agent. It is important to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared by adding this thickening agent from a solution.

Pharmaceutically acceptable preservatives or cell stabilizers may be employed to enhance the longevity of the MAPC composition. If such preservatives are included, it is well within the skill of the artisan to select a composition that does not affect the viability or efficacy of the MAPCs.

Those skilled in the art will recognize that the components of the composition should be chemically inert. This is not a problem for those skilled in the art who are familiar with the principles of chemistry and medicine. Problems can be easily avoided by reference to authoritative textbooks or by simple experiments (not involving excessive experimentation) using the information provided in this disclosure and the literature cited herein or information readily available in the art.

Sterile injectable solutions can be prepared by incorporating the cells used to practice the present invention in the required amount of an appropriate solvent along with varying amounts of the other ingredients as required.

In some embodiments, MAPCs are formulated into unit dose injectable forms, such as solutions, suspensions, or emulsions. Pharmaceutical preparations suitable for MAPCs injection are typically sterile aqueous solutions and dispersants. The carrier of the injectable preparation can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and a suitable mixture thereof.

The technical staff can easily determine the amount of cell and optional additive, medium and/or carrier in the composition for being administered by the inventive method.Usually, any additive (except cell) is 0.001-50wt% in solution as in phosphate buffered saline (PBS).The amount of active component is the scope of microgram amount to milligram amount, as approximately 0.0001 to approximately 5wt%, preferably approximately 0.0001 to approximately 1wt%, most preferably approximately 0.0001 to approximately 0.05wt% or approximately 0.001 to approximately 20wt%, preferably approximately 0.01 to approximately 10wt%, most preferably approximately 0.05 to approximately 5wt%.

For any composition to be administered to an animal or human and for any particular method of administration, it is preferred to determine: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent such as a mouse or rat); and the dosage of the composition, the concentration of the components thereof, and the timing of administration of the composition that will elicit an appropriate response. This determination can be made without undue experimentation, based on the knowledge of the skilled artisan, this disclosure, and the literature cited herein. Similarly, the timing of sequential administration can be determined without undue experimentation.

In some embodiments, MAPCs are encapsulated for administration, particularly where encapsulation improves the effectiveness of therapy or provides handling and/or shelf life benefits. In some embodiments where encapsulation improves the efficacy of MAPC-mediated immunosuppression, inclusion can also thereby reduce the need for immunosuppressive drug therapy.

Moreover, in some embodiments, the encapsulation can provide a barrier to the subject's immune system, which can further reduce the response of the subject's immune system to the MAPCs (MAPCs are generally non-immunogenic or only weakly immunogenic in allogeneic transplants), thereby reducing any transplant rejection or inflammation that may occur when the cells are administered.

In various embodiments in which MAPCs are administered in admixture with another type of cell (which is typically more immunogenic in an allogeneic or xenogeneic environment), encapsulation can reduce or eliminate the host's adverse immune response to the non-MAPC cells and/or GVHD that may occur in an immunocompromised host if the admixed cells were immunologically active and recognized the host as non-self.

MAPCs can be encapsulated in membranes and capsules prior to implantation. It is contemplated that any of a number of existing cell encapsulation methods can be employed. In some embodiments, cells are encapsulated individually. In some embodiments, multiple cells are encapsulated within the same membrane. In embodiments where cells are to be removed after implantation, the relatively large size of the structure encapsulating multiple cells (e.g., within a single membrane) can provide a convenient means for recovery.

A variety of materials can be used to microencapsulate MAPCs in various embodiments. Such materials include, for example, polymer capsules, alginate-poly-L-lysine-alginate microcapsules, poly-L-lysine barium alginate capsules, barium alginate capsules, polyacrylonitrile/polyvinyl chloride (PAN/PVC) hollow fibers, and polyethersulfone (PES) hollow fibers.

Cell microencapsulation techniques that can be used to administer MAPCs are well known to those skilled in the art and are described, for example, in Chang, P., et al., 1999; Matthew, H.W., et al., 1991; Yanagi, K., et al., 1989; Cai Z.H., et al., 1988; Chang, T.M., 1992 and in U.S. Pat. No. 5,639,275 (which describes, for example, biocompatible capsules for long-term maintenance of cells capable of stably expressing biologically active molecules). Additional encapsulation methods are described in European Patent Publication No. 301,777 and U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272; 5,578,442; 5,639,275 and 5,676,943. The contents of all aforementioned documents and patents regarding encapsulation of MAPCs are incorporated herein by reference.

Certain embodiments incorporate MAPCs into polymers such as biopolymers or synthetic polymers. Examples of biopolymers include, but are not limited to, fibronectin, fibrinogen, thrombin, collagen, and proteoglycans. Other factors, such as the above-mentioned cytokines, may also be incorporated into polymers. In other embodiments of the present invention, MAPCs may be incorporated into the voids of a three-dimensional gel. Large polymers or gels typically require surgical implantation. Polymers or gels that can be formulated into sufficiently small particles or fibers may be administered through other common, more convenient non-surgical approaches.

The pharmaceutical compositions of the present invention can be prepared in a variety of forms, including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes, as well as other sustained-release formulations such as shaped polymeric gels. Oral liquid pharmaceutical compositions can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or can be presented as a dry product for reconstitution with water or other suitable media before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifiers, non-aqueous media (which may include edible oils), or preservatives. Oral dosage forms can be formulated so that the cells are released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and the references contained therein.

Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solutions and other materials commonly used in the art.

For administration by inhalation, the cells can be conveniently delivered from an insufflator, nebulizer, or pressurized pack or other convenient spray delivery vehicle. The pressurized pack may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, administration may take the form of a dry powder composition, for example a powder mix of the modulator and a suitable powder base such as lactose or starch. The powder composition may be provided in unit dosage form, for example in capsules or cartridges or, for example, in gelatin or blister packs from which the powder can be administered with the aid of an inhaler or insufflator. For intranasal administration, the cells may be administered by liquid spray, such as from a plastic bottle sprayer.

Other active ingredients

MAPCs can be administered with other pharmaceutically active agents. In some embodiments, one or more such agents are formulated together with MAPCs for administration. In some embodiments, MAPCs and the one or more agents are in separate formulations. In some embodiments, the compositions comprising MAPCs and/or the one or more agents are formulated to assist in the administration of the compositions.

MAPCs can be administered in a formulation containing an immunosuppressant, such as a corticosteroid, cyclosporine A, a cyclosporine-like immunosuppressant, cyclophosphamide, antithymocyte globulin, azathioprine, rapamycin, FK-506, and any combination of any of the macrolide-like immunosuppressants other than FK-506 and rapamycin. In certain embodiments, the agent comprises a corticosteroid, cyclosporine A, azathioprine, cyclophosphamide, rapamycin, and/or FK-506. The aforementioned immunosuppressant may be solely such additional agent, or may be combined with other agents such as those mentioned herein. Other immunosuppressants include tacrolimus, mycophenolate mofetil, and sirolimus.

Such agents also include antibiotic agents, antifungal agents, and antiviral agents, to name just a few of the other pharmacologically active substances and compositions that may be used in accordance with embodiments of the present invention.

Typical antibiotic or antifungal compounds include, but are not limited to, penicillin, streptomycin, amphotericin, ampicillin, gentamicin, kanamycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, zeocin, and cephalosporins, aminoglycosides, and echinocandins.

More of these additives are related to the fact that MAPCs, like other stem cells, will "home" to an environment that is conducive to their growth and function after being administered to a subject. This "homing" often concentrates the cells in areas where they are needed, such as areas of immune disorders, dysfunction or disease. There are a variety of substances known to stimulate homing. They include growth factors and nutrient signaling agents such as cytokines. They can be used to promote the homing of MAPCs to the target site of treatment. They can be administered to the subject before treatment with MAPCs, together with MAPCs, or after administration of MAPCs.

Certain cytokines, for example, can alter or influence the migration of MAPCs or their differentiated cells to sites in need of treatment, such as sites of immunodeficiency. Cytokines that can be used in this regard include, but are not limited to, stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), angiopoietin-1, placenta-derived growth factor (PIGF), granulocyte colony-stimulating factor (G-CSF), cytokines that can stimulate the expression of endothelial adhesion molecules such as ICAMs and VCAMs, and cytokines that can cause or promote homing.

They can be administered to subjects as a pre-treatment, together with MAPCs, or after administration of MAPCs to promote homing to the desired site and to achieve improved therapeutic effects through improved homing or through other mechanisms. Such factors can be combined with MAPCs in a formulation suitable for administering them together. Alternatively, such factors can be formulated and administered separately.

The order of administration, formulation, dosage, frequency of administration, and route of administration of each factor (such as the above-mentioned cytokines) and MAPCs will generally vary depending on the disorder or disease being treated and its severity, the subject, other therapies being administered, the stage of the disorder or disease, and prognostic factors, etc. General protocols established for other therapeutics can provide a framework for determining appropriate dosages in direct or adjuvant therapies mediated by MAPCs. This information, along with the additional information provided herein, will enable a skilled artisan to determine appropriate administration procedures according to the embodiments of the present invention without undue experimentation.

way

MAPCs can be administered to a subject by any of a variety of routes known to those skilled in the art for administering cells to a subject.

Methods that can be used in various embodiments of the present invention in this regard include methods of administering MAPCs by parenteral routes. Parenteral routes of administration that can be used in various embodiments of the present invention include administration by intravenous, intraarterial, intracardial, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intradermal, intradermal, subcutaneous, and/or intramuscular injection. In some embodiments, intravenous, intraarterial, intradermal, intradermal, subcutaneous, and/or intramuscular injection is used. In some embodiments, intravenous, intraarterial, intradermal, subcutaneous, and/or intramuscular injection is used.

In various embodiments of the present invention, MAPCs are administered by systemic injection. Systemic injection, such as intravenous injection, provides one of the simplest and least invasive approaches to administering MAPCs. In some cases, these approaches may require high MAPC dosages to achieve optimal effectiveness and/or homing of MAPCs to the target site. In various embodiments, MAPCs can be administered by targeted and/or local injection to ensure optimal effect at the target site.

In some embodiments of the present invention, MAPCs can be administered to a subject via a hypodermic needle via a syringe. In various embodiments, MAPCs are administered to a subject via a catheter. In multiple embodiments, MAPCs are administered by surgical implantation. In addition, in this regard, in various embodiments of the present invention, MAPCs are administered to a subject by implantation via an arthroscopic procedure. In some embodiments, MAPCs are administered to a subject in or on a solid carrier (such as a polymer or gel). In various embodiments, MAPCs are administered to a subject in encapsulated form.

In additional embodiments of the invention, MAPCs are suitably formulated for oral, rectal, epidermal, ocular, nasal, and/or pulmonary delivery and administered accordingly.

dose

The composition can be administered in dosages and techniques known to those skilled in the medical and veterinary arts, taking into account factors such as the age, sex, weight, and physical condition of the particular patient and the formulation to be administered (e.g., whether it is a solid formulation or a liquid formulation). The skilled person, based on this disclosure, the literature cited herein, and the knowledge in the art, can determine the dosage for humans or other mammals without undue experimentation.

The dosage of MAPCs suitable for use in accordance with the various embodiments of the present invention depends on a number of factors. This dosage can vary greatly from one situation to another. Parameters that determine the optimal dosage of MAPCs for primary and adjunctive therapy typically include some or all of the following: the disease being treated and its stage; the species of the subject; their health, sex, age, weight, and metabolic rate; the subject's immune activity; other therapies being administered; and potential complications expected from the subject's medical history or genotype. Parameters may also include: whether the MAPCs are syngeneic, autologous, allogeneic, or xenogeneic; their efficacy (specific activity); the site and/or distribution that must be targeted in order for the MAPCs to be effective; and characteristics of that site such as accessibility to the MAPCs and/or the engraftment of the MAPCs. Additional parameters include the co-administration of MAPCs with other factors, such as growth factors and cytokines. The optimal dosage in a given situation also takes into account how the cells are formulated, how the cells are administered, and the extent to which the cells will be localized at the target site after administration. Ultimately, the optimal dose must be determined to provide an effective dose that is neither below the threshold for maximal beneficial effects nor above the threshold at which the dose of MAPCs is accompanied by adverse effects that outweigh the benefits of the increased dose.

For some embodiments, the optimal dose of MAPCs is within the range of doses used for autologous mononuclear bone marrow transplantation. For fairly pure MAPCs preparations, the optimal dose for each administration in various embodiments is in the range of 10 ⁴ -10 ⁸ MAPC cells/kg recipient mass. In some embodiments, the optimal dose for each administration is between 10 ⁵ -10 ⁷ MAPC cells/kg. In many embodiments, the optimal dose for each administration is between 5x 10 ⁵ -5x 10 ⁶ MAPC cells/kg. For reference, the higher doses in the aforementioned dosage ranges are similar to the doses of nucleated cells used in autologous mononuclear bone marrow transplantation. Some lower doses are similar to the number of CD34 ⁺ cells/kg used in autologous mononuclear bone marrow transplantation.

It will be appreciated that a single dose may be delivered all at once, in divided doses, or continuously over a period of time. The entire dose may also be delivered to a single site, or distributed over several sites.

In various embodiments, MAPCs can be administered in an initial dose and then maintained by further administration of MAPCs. MAPCs can be initially administered by one method and then administered by the same method or one or more different methods. The MAPCs level of the subject can be maintained by continuously administering the cells. In various embodiments, MAPCs are either initially administered by intravenous injection, or their levels in the subject are maintained, or both. In various embodiments, other modes of administration are used, depending on the patient's physical condition and other factors discussed elsewhere herein.

It should be noted that treatment times for human subjects are generally longer than for experimental animals, but the length of treatment is generally commensurate with the length of the disease process and the effectiveness of the treatment. Those skilled in the art will take this into account when determining appropriate dosages for humans using results from other procedures performed in humans and/or in animals (e.g., rats, mice, non-human primates, etc.). Such determinations based on these considerations and in consideration of the guidance provided by this disclosure and the prior art will enable the skilled person to do so without undue experimentation.

Suitable regimens for the initial administration and further doses, or suitable regimens for sequential administration, may all be the same or may be different. A skilled person can determine the appropriate regimen based on this disclosure, the literature cited herein, and the knowledge in the art.

The dosage, frequency, and duration of treatment will depend on many factors, including the nature of the disease, the subject, and other therapies that may be administered. Thus, there are a variety of regimens available for administering MAPCs.

In some embodiments, MAPCs are administered to a subject in a single dose. In other embodiments, MAPCs are administered to a subject in a series of two or more doses. In some other embodiments, in which MAPCs are administered in a single dose, in two doses, and/or in more than two doses, the doses may be the same or different and administered at equal or unequal intervals.

MAPCs can be administered multiple times over a variety of timeframes. In some embodiments, MAPCs are administered in less than a day. In other embodiments, they are administered in two, three, four, five, or six days. In some embodiments, MAPCs are administered once or multiple times per week over a period of several weeks. In other embodiments, they are administered over a period of several weeks, lasting from one month to several months. In various embodiments, they can be administered over a period of several months. In other embodiments, they can be administered over a period of one or more years. Typically, the length of treatment will be commensurate with the length of the disease process, the effectiveness of the therapy being applied, and the physical condition and response of the subject being treated.

Therapeutic uses of immunomodulatory MAPCs

The immunomodulatory properties of MAPCs can be used to treat a variety of disorders, dysfunctions, and diseases, such as those that present deleterious immune system processes and effects, either secondary in nature or as a side effect of treatment. Several examples are discussed below.

In this regard, many embodiments relate to giving MAPCs to a subject whose immune system is weakened (or defective) as sole therapy or as an adjuvant therapy with another treatment. In this regard, in multiple embodiments of the present invention, MAPCs are assisted in the radiotherapy or chemotherapy or the combination of radiotherapy and chemotherapy that has been given, is being given, or will be given to the subject to give the subject. In many of these embodiments, radiotherapy, chemotherapy or the combination of radiotherapy and chemotherapy is a part of transplantation therapy. In multiple embodiments, MAPCs are given to treat harmful immune responses such as HVG or GVHD.

In this regard, in various embodiments, the subject is the recipient of a non-syngeneic (typically allogeneic) platelet or bone marrow cell transplant, the subject's immune system has been weakened or ablated by radiation therapy, chemotherapy, or a combination of radiation therapy and chemotherapy, the subject is being administered immunosuppressive drugs, the subject is at risk of developing graft-versus-host disease or has already developed the disease, and MAPCs are administered to the subject in adjunct to any one or more of the transplant, radiation therapy and/or chemotherapy and immunosuppressive drugs to treat, such as to ameliorate, prevent, or eliminate, the subject's graft-versus-host disease.

tumor

The term "tumor" generally refers to diseases involving the clonal proliferation of cells. Tumors can be benign, meaning they are non-progressive and non-recurrent and, if so, are generally not life-threatening. Tumors can also be malignant, meaning they progressively worsen and spread and, therefore, are generally life-threatening and are often fatal.

In various embodiments, MAPCs are administered to a subject suffering from a tumor to assist in the treatment of the tumor. For example, in this regard, in some embodiments of the present invention, the subject is at risk of or is suffering from a tumor of blood or bone marrow cells and has undergone or will undergo a blood or bone marrow transplant. Using the MAPC separation, characterization, and amplification methods described herein, combined with the disclosure herein regarding the immunosuppressive properties of MAPCs, MAPCs can be administered to treat, such as prevent, inhibit, or reduce, adverse immune responses such as HVG and GVHD that can complicate transplantation therapy.

In a number of embodiments relating to transplantation therapy, MAPCs can be used for immunosuppressive purposes alone or in combination with other agents. MAPCs can be given before, during, or after one or more transplants are performed. If given during a transplant, MAPCs can be given separately from the transplant material or together. If given separately, MAPCs can be given sequentially or simultaneously with other transplant materials. In addition, in addition to or in lieu of giving MAPCs while or about the same time as a transplant is given, MAPCs can be given well before and/or well after the transplant.

Other agents that can be used in combination with MAPCs, particularly in transplantation therapy, include immunomodulators, such as those described elsewhere herein, particularly immunosuppressive agents, more particularly immunosuppressive agents described elsewhere herein, particularly for this purpose, namely, one or more of corticosteroids, cyclosporine A, cyclosporine-like immunosuppressive compounds, azathioprine, cyclophosphamide, methotrexate, and immunosuppressive monoclonal antibody agents.

In this regard, myeloid neoplastic diseases treated with MAPCs in embodiments of the present invention include myeloproliferative disorders ("MPDs"), myelodysplastic syndromes (or states) ("MDSs"), leukemias, and lymphoproliferative disorders including multiple myeloma and lymphoma.

MPDs are characterized by abnormal and spontaneous proliferation of cells in the blood and marrow. The disease may involve only one cell type or several cell types. Typically, MPDs involve three cell lineages: the erythroid lineage, the granulocyte lineage, and the platelet lineage. The involvement of these three lineages varies with each MPD and with each type that occurs. Generally, they are affected differently, with one cell lineage being predominantly affected in a given tumor. MPDs are not overtly malignant; however, they are classified as tumors and are characterized by abnormal self-replication of hematopoietic precursor cells in the blood and marrow. Despite this, MPDs have the potential to develop into acute leukemias.

Like MPDs, MDSs are clonal disorders characterized by abnormal self-replication of hematopoietic precursor cells in the blood and bone marrow. Like MPDs, they can develop into acute leukemias. Most, but not all, MDSs (except in cases of chronic myelomonocytic leukemia) exhibit peripheral blood cytopenias, whereas MPDs do not.

The laboratory and clinical manifestations of these diseases can vary over their course and between episodes. The manifestations may overlap, and a reliable diagnosis distinguishing one disease from all others can be difficult. Therefore, the diagnosis of tumors of the hematopoietic cells of the bone marrow requires particular caution to avoid misdiagnosing a benign condition that is actually a fatal malignancy.

The following diseases are myeloproliferative disorders (MPDs) that can be treated primarily or adjunctively with MAPCs in various embodiments of the present invention: chronic myeloid leukemia ("CML")/chronic granulocytic leukemia ("CGL"), agnogenic myelofibrosis, essential thrombocythemia, and polycythemia vera.

The following diseases are myelodysplastic syndromes (MDSs) that can be treated primarily or adjunctively with MAPCs in various embodiments of the present invention: refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, refractory anemia with excess transforming blasts, and chronic myelomonocytic leukemia.

The following diseases are among the lymphoproliferative disorders (including multiple myeloma and lymphoma) that can be treated primarily or adjunctively with MAPCs in various embodiments of the present invention: pre-B acute lymphoblastic leukemia, chronic lymphocytic leukemia ("CLL"), B cell lymphoma, hairy cell leukemia, myeloma, multiple myeloma, T acute lymphoblastic leukemia, peripheral T cell lymphoma, other lymphoid leukemias and other lymphomas.

In various embodiments of the present invention, tumors that can be treated primarily or adjunctively with MAPCs include: benign tumors of bone marrow cells, myeloproliferative diseases, myelodysplastic syndromes or acute leukemias; chronic myelogenous leukemia ("CML") (also known as chronic myeloid leukemia ("CGL")), unexplained myelofibrosis, essential thrombocythemia, polycythemia vera, other myeloproliferative diseases, acute multiple myeloma, myeloblastic leukemia, acute promyelocytic leukemia, pre-B acute lymphoblastic leukemia, chronic lymphocytic leukemia ("CLL"), B cell lymphoma, hairy cell leukemia, myeloma, T acute lymphoblastic leukemia, peripheral T cell lymphoma, other lymphoid leukemias, other lymphomas or other acute leukemias.

MAPCs may be administered adjunctively in the treatment of any of the aforementioned diseases.

Treatment involving immune ablation or deficiency

Acute leukemias are often difficult to treat with therapies that are effective for other malignancies. This is partly because cells, including tumor cells, migrate from the bone marrow. Partly because the bone marrow is dispersed throughout the bones. And partly, undoubtedly, because of the characteristics of the cells themselves and their transformation.

Currently, the standard treatment for hematological malignancies involves removing all of a patient's blood-forming cells. This is impossible without also removing the patient's healthy blood-forming cells. Typically, patients are treated with chemoradiation at doses high enough to kill nearly all of their bone marrow cells, both normal and tumor cells. The side effects of this treatment are severe, and its impact on the patient is unpleasant, painful, and physically and emotionally exhausting. The treatment not only removes diseased tissue and cells, it also removes the patient's blood-forming and immune systems. The treatment leaves patients immunocompromised, dependent on transfusions, and so highly susceptible to infection that even a mild exposure to an infectious agent can be fatal.

Normal hematopoietic capacity is then restored by an autologous or allogeneic peripheral blood or bone marrow transplant. Unfortunately, the patient's immune system is severely compromised not only by the ablation treatment but also, in the case of an allogeneic transplant, by the intentional immunosuppression that is required to prevent rejection of the graft and to ensure the engraftment and proliferation of new hematopoietic stem cells that will repopulate the patient's bone marrow and regenerate the patient's hematopoietic and immune cells.

Many complications are encountered when performing such transplants to regenerate the hematopoietic and immune systems in immunocompromised hosts. One complication is rejection caused by residual immunocompetent cells and processes in the host, referred to herein as an HVG reaction. Another complication is caused by immunocompetent cells in the graft, referred to herein as GVHD.

These complications can be avoided by using either syngeneic or autologous donor material. However, syngeneic donors are generally rare, and autologous transplants carry a high risk of disease recurrence. Therefore, transplants typically use allogeneic cells and tissues obtained from HLA-compatible donors. Unfortunately, this procedure can lead to GVHD, which occurs in mild or severe forms in the majority of patients receiving this form of therapy. If not at least ameliorated, these immune reactions can lead to the failure of the transplant and can themselves be fatal to the patient.

A variety of agents have been developed to suppress immune responses, thereby improving transplant complications such as HVG and GVHD, as described above. In some transplant therapies, such as bone marrow and peripheral blood transplants, there are agents that are sufficiently effective to reduce adverse immune responses to manageable levels. These agents have improved the prognosis of transplant patients to some extent, but none of them is completely effective, and all of them have considerable disadvantages.

It has been found (described in more detail elsewhere herein) that MAPCs do not elicit an immune response in allogeneic hosts. Therefore, transplantation of MAPCs into allogeneic hosts does not result in allogeneic graft rejection (ie, HVG).

Furthermore, it was found that allogeneic MAPCs could be administered to the host at high concentrations without adversely affecting respiration.

It has also been found (which is described in more detail elsewhere herein) that MAPCs can modulate immune responses. Specifically in this regard, MAPCs have been found to suppress immune responses, including but not limited to those involving, for example, HVG reactions and GVHD (to name two examples). More specifically in this regard, MAPCs have been found to suppress T cell proliferation even in the presence of potent T cell stimulators such as concanavalin A and allogeneic or xenogeneic stimulator cells.

Furthermore, it was found that even relatively small amounts of MAPCs could suppress these responses. Indeed, just 3% MAPCs were sufficient to reduce T cell responses in vitro by 50% in a mixed lymphocyte reaction.

Thus, embodiments of the present invention provide, among other things, compositions and methods for treating, eg, ameliorating and/or remediating or eliminating, tumors, such as tumors of hematopoietic cells, particularly tumors of the bone marrow.

These neoplasms include myeloproliferative disorders (MPDs) such as chronic myeloid leukemia (also called "chronic granulocytic leukemia" and "CGL"), myelofibrosis of unknown cause, essential thrombocythemia, and polycythemia vera; myelodysplastic syndromes (MDSs) such as refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, refractory anemia with excess transformed blasts, and chronic myelomonocytic leukemia; and overtly malignant neoplasms—acute leukemias such as acute myeloblastic leukemia, chronic myeloid leukemia (CML), acute promyelocytic leukemia, B-acute lymphoblastic leukemia, CLL, B-cell lymphoma, hairy cell leukemia, myeloma, T-acute lymphoblastic leukemia, peripheral T-cell lymphoma, and other lymphoid leukemias and lymphomas.

MAPCs may be administered adjunctively in the treatment of any of the aforementioned diseases.

Anemia and other blood disorders

In various embodiments of the present invention, MAPCs can be used to treat anemia or other blood diseases, often in an adjuvant manner. Various embodiments in this regard are embodiments in which MAPCs are used to treat the following anemias and/or blood diseases alone, primarily, or adjuvantly: hemoglobinopathies, thalassemias, bone marrow failure syndromes, sickle cell anemia, aplastic anemia, or immune hemolytic anemia. Diseases also include refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excessive primitive cells, refractory anemia with excessive transforming primitive cells, chronic myelomonocytic leukemia or other myelodysplastic syndromes, and in some embodiments, Fanconi anemia.

MAPCs may be administered adjunctively in the treatment of any of the aforementioned diseases.

immune diseases

Embodiments of the present invention relate to the use of MAPC immunomodulation, alone or as an adjunct therapy, to treat immune dysfunctions, disorders, or diseases. Embodiments in this regard relate to congenital immunodeficiencies and autoimmune dysfunctions, disorders, and diseases. Various embodiments in this regard relate to the use of MAPCs to treat, alone or adjunctively, Crohn's disease, Guillain-Barré syndrome, lupus erythematosus (also known as "SLE" and systemic lupus erythematosus), multiple sclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, diabetes mellitus (type I), Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome ("APS"), opsoclonus-myoclonus syndrome ("OMS"), temporal arteritis, acute disseminated encephalomyelitis ("ADEM" and "ADE"), Goodpasture's syndrome, Wegener's granulomatosis, celiac disease, pemphigus, polyarthritis, and warm autoimmune hemolytic anemia.

Specific embodiments within these embodiments relate to Crohn's disease, lupus erythematosus (also known as "SLE" and systemic lupus erythematosus), multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease, diabetes mellitus (type I), Reiter's syndrome, primary biliary cirrhosis, celiac disease, polyarthritis, and warm autoimmune hemolytic anemia.

Additionally, in this regard, MAPCs may be used in various embodiments solely, primarily, or adjunctively to treat a variety of diseases believed to have an autoimmune component, including but not limited to embodiments for the treatment of endometriosis, interstitial cystitis, neuromyotonia, scleroderma, progressive systemic scleroderma, vitiligo, vulvodynia, Chagas' disease, sarcoidosis, chronic fatigue syndrome, and dysautonomia.

Genetic immune system diseases include severe combined immunodeficiency (SCID), including but not limited to SCID with adenosine deaminase deficiency (ADA-SCID), X-linked SCID, SCID without T cells and B cells, SCID without T cells, normal B cells, Omenn syndrome, neutropenia, including but not limited to Kostmann syndrome, Myelokathexis; ataxia telangiectasia, bare lymphocyte syndrome, common variable immunodeficiency, DiGeorge syndrome, leukocyte adhesion deficiency; and phagocyte diseases (phagocytes are cells of the immune system that can engulf and kill foreign organisms), including but not limited to Chediak-Higashi syndrome, chronic granulomatous disease, neutrophil actin deficiency, and reticulum dysplasia.

MAPCs may be administered adjunctively in the treatment of any of the aforementioned diseases.

Inflammatory diseases

Additionally, in various embodiments of the present invention, MAPCs may be used as the sole agent or adjunctively to treat inflammatory diseases. In many such embodiments, MAPCs may be used to treat severe inflammatory conditions, such as those caused by acute allergic reactions or as a secondary component of other diseases or treatments. The use of MAPCs in this regard is currently limited primarily to acute situations in which the subject is in danger of significant disability or loss of life.

MAPCs may be administered adjunctively in the treatment of any of the aforementioned diseases.

MAPCs described in U.S. Patent 7,015,037

Human MAPCs are described in the art. Methods for separating human and mouse MAPCs are well known in the art. Therefore, bone marrow aspirates, brain or liver biopsies, and other organs can now be obtained by those skilled in the art, and these cells can be separated by relying on genes expressed (or not expressed) in these cells (e.g., by functional or morphological determinations, as disclosed in the applications cited above, which have been incorporated herein by reference). This method is described in, for example, U.S. Patent No. 7,015,037, which is incorporated herein by reference for its description of MAPCs and preparation methods.

Isolation and growth of MAPCs as described in PCT/US00/21387

Methods for separating human and mouse MAPCs are well known in the art. They are described in, for example, U.S. Patent No. 7,015,037, PCT/US00/21387 (announced with WO 01/11011) and PCT/US02/04652 (announced with WO 02/064748), and these methods, together with the characterization of MAPCs disclosed in these patents, are incorporated herein by reference.

MAPCs were originally isolated from bone marrow, but have since been established from other tissues, including brain and muscle (Jiang, Y. et al., 2002). Thus, MAPCs can be isolated from a variety of sources, including bone marrow, placenta, umbilical cord and cord blood, muscle, brain, liver, spinal cord, blood, or skin. For example, MAPCs can be derived from bone marrow aspirates, which can be obtained by standard methods available to those skilled in the art (see, for example, Muschler, G.F., et al., 1997; Batinic, D., et al., 1990).

Phenotype of human MAPCs under the conditions described in U.S. Patent 7,015,037

Immunophenotypic analysis of human MAPCs obtained after 22-25 cell doublings by FACS showed that the cells could not express CD31, CD34, CD36, CD38, CD45, CD50, CD62E and -P, HLA-DR, Muc18, STRO-1, cKit, Tie/Tek; they could express low levels of CD44, class I HLA and β2-microglobulin, but could express CD10, CD13, CD49b, CD49e, CDw90, and Flk1 (N>10).

Once the cells had undergone more than 40 doublings in culture, seeded at approximately 2 x 10 ³ /cm ² , the phenotype became more homogeneous, with no cells expressing class I HLA or CD44 (n=6). When the cells were grown at a higher confluence, they expressed high levels of Mucl8, CD44, class I HLA, and β2-microglobulin, similar to the phenotype described for MSCs (N=8) (Pittenger, 1999).

Immunohistochemistry showed that human MAPCs grown at a seeding density of approximately 2 x 10 ³ /cm ² expressed EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and -B, and a small population (1-10%) of MAPCs were stained by anti-SSEA4 antibody (Kannagi, R, 1983).

Using Clontech cDNA arrays, we determined the gene expression profiles of human MAPCs cultured at a seeding density of approximately 2 x 10 ³ cells/cm ² for 22-26 cell doublings:

A. MAPCs do not express CD31, CD36, CD62E, CD62P, CD44-H, cKit, Tie, IL1 receptor, IL3 receptor, IL6 receptor, IL11 receptor, G CSF, GM-CSF, Epo, Flt3-L or CNTF, but express low levels of class I HLA, CD44-E and Muc-18 mRNA.

B. MAPCs express mRNA of cytokines BMP1, BMP5, VEGF, HGF, KGF, and MCP1; cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130, LIF-R, activator proteins-R1 and -R2, TGFR-2, and BMP-R1A; and adhesion receptors CD49c, CD49d, CD29, and CD10.

MAPCs express mRNA for hTRT and TRF1; POU domain transcription factors oct-4 and sox-2 (oct-4 is required to maintain the undifferentiated state of ES/ECs, Uwanogho D., 1995), sox 11 (neural development), sox 9 (chondrogenesis) (Lefebvre V., 1998); homeotic domain transcription factors: Hox-a4 and -a5 (cervical and thoracic skeleton specification; respiratory tract organogenesis) (Packer AI, 2000), Hox-a9 (myelopoiesis) (Lawrence H, 1997), Dlx4 (specification of the forebrain and surrounding structures of the head) (Akimenko MA, 1994), MSX1 (embryonic mesoderm, adult heart and muscle, chondrogenesis and osteogenesis) (Foerst-Potts L. 1997), PDX1 (pancreas) (Offield MF, 1996).

D. RT-PCR confirmed the presence of oct-4, LIF-R and hTRT mRNA.

In addition, RT-PCR showed that rex-1 mRNA and rox-1 mRNA were expressed in MAPCs.

Oct-4, rex-1 and rox-1 are expressed in MAPCs derived from human and mouse bone marrow and MAPCs derived from mouse liver and brain. Human MAPCs express LIF-R and stain positive for SSEA-4. Finally, it was found that the levels of oct-4, LIF-R, rex-1 and rox-1 mRNA increased in human MAPCs cultured for more than 30 cell doublings, which resulted in the production of phenotypically more homogeneous cells. In contrast, MAPCs cultured at high density lost the expression of these markers. This is related to aging and the loss of the ability to differentiate into cells other than chondrocytes, osteoblasts and adipocytes before 40 cell doublings. Therefore, the presence of oct-4, together with rex-1, rox-1 and sox-2, is associated with the presence of most primitive cells in MAPCs culture.

Methods for culturing MAPCs are well known in the art. (See, for example, U.S. Patent No. 7,015,037, which is incorporated herein by reference for methods of culturing MAPCs). The density of culturing MAPCs can vary from about 100 cells/ ^cm or about 150 cells/ ^cm to about 10,000 cells/ ^cm , including from about 200 cells/ ^cm to about 1500 cells/ ^cm to about 2000 cells/ ^cm . Density can be different for different species. In addition, the optimal density can vary with culture conditions and cell source. Determining the optimal density for a given set of culture conditions and cells is within the skill of the technician.

Also, effective atmospheric oxygen concentrations of less than about 10%, including about 3-5%, may be used at any time during the isolation, growth, and differentiation of MAPCs in culture.

The invention is further described by the following illustrative non-limiting examples.

Example

Example 1: Human MAPCs (from bone marrow mononuclear cells)

Bone marrow mononuclear cells were obtained from bone marrow aspirates of the posterior iliac spine from >80 healthy human volunteers. 10-100 cubic centimeters of bone marrow were obtained from each subject. Mononuclear cells ("MNCs") were isolated by centrifugation of the bone marrow through a Ficoll-Paque density gradient (Sigma Chemical Co., St. Louis, MO). Bone marrow MNCs were incubated with CD45 and glycophorin A microbeads (Miltenyi Biotec, Sunnyvale, CA) for 15 minutes, and the samples were placed in front of a SuperMACS magnet to remove CD45 ⁺ GlyA ⁺ cells. The eluted cells were 99.5% ^{CD45- GlyA-} .

Depletion of CD45 ⁺ GlyA ⁺ cells resulted in the recovery of CD45 ^{- GlyA-} cells, which comprised approximately 0.05-0.10% of total bone marrow mononuclear cells. Cells were selected that did not express the common leukocyte antigen CD45 or the erythroid precursor marker glycophorin A (GlyA). CD45 ^{- GlyA-} cells comprised ^1/103 of bone marrow mononuclear cells. CD45 ^{- GlyA-} cells were plated in wells coated with fibronectin in 2% FCS, EGF, PDGF-BB, dexamethasone, insulin, linoleic acid, and ascorbic acid. After 7-21 days, small clusters of adherent cells developed. Using limiting dilution assays, the frequency of cells giving rise to these adherent cell clusters was 1/5 x ¹⁰³ CD45 ^{- GlyA-} cells. When cell colonies appeared (approximately 10 ³ cells), cells were recovered by trypsinization and re-seeded at a 1:4 dilution every 3-5 days under the same culture conditions. The cell density was maintained between 2-8 x 10 ³ cells/cm ² .

Example 2: Mouse MAPCs

All tissues were obtained according to the guidelines of the University of Minnesota IACUC. BM mononuclear cells (BMMNCs) were obtained by Ficoll Hypaque separation. BM was obtained from 5-6 week old ROSA26 mice or C57/BL6 mice. Alternatively, muscle and brain tissue were obtained from 3 day old 129 mice. The muscles of the proximal front and rear limbs were removed and thoroughly minced. The tissue was treated with 0.2% collagenase (Sigma Chemical Co., St Louis, MO) at 37°C for 1 hour, followed by 0.1% trypsin (Invitrogen, Grand Island, NY) for 45 minutes. The cells were then vigorously ground and passed through a 70 μm filter. The cell suspension was collected and centrifuged at 1600 rpm for 10 minutes. The brain tissue was dissected and thoroughly minced. The cells were dissociated by incubation at 37°C (with 0.1% trypsin and 0.1% DNase Sigma) for 30 minutes. The cells were then vigorously ground and passed through a 70 μm filter. The cell suspension was collected and centrifuged at 1600 rpm for 10 minutes.

BMMNCs or muscle or brain suspensions were seeded at 1 x 10 ⁵ /cm ² in expansion medium [2% FCS in low-glucose Dulbecco's Minimal Essential Medium (LG-DMEM), 10 ng/ml each of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF)] and maintained at 5 x 10 ³ /cm ^2. After 3-4 weeks, cells were recovered using trypsin/EDTA, and CD45 ⁺ GlyA ⁺ cells were removed using micromagnetic beads. The resulting CD45 ^- GlyA ^- cells were seeded at 10 cells/well in FN-coated 96-well plates and expanded to a cell density of 0.5-1.5 x 10 ³ /cm ^2. Regardless of the tissue from which MAPCs were derived, their expansion potential was similar.

Example 3: Rat MAPCs

BM and MNCs were obtained from Sprague Dawley, Lewis, or Wistar rats and plated under conditions similar to those described for mMAPCs.

Example 4: Clonability of Mouse and Rat MAPCs

To demonstrate that the differentiated cells were single-cell derived and that MAPCs were indeed "clonal" pluripotent cells, cultures were generated in which MAPCs had been transduced with a retroviral vector. Undifferentiated MAPCs and their progeny were found to have the retroviral insertion at the same site in the genome.

Studies were conducted using two independently derived ROSA26 MAPCs, two C57BL/6 MAPCs, and an rMAPC population expanded from 40 to greater than 90 PD, as well as "clonal" mice and "clonal" rMAPCs transduced with eGFP. No differences were observed between eGFP-transduced and non-transduced cells. Notably, eGFP expression persisted in differentiated MAPCs.

Specifically, mouse and rat BMMNCs cultured on FN for three weeks with EGF, PDGF-BB and LIF were transduced with eGFP oncoretroviral vectors on two consecutive days. CD45 ⁺ and GlyA ⁺ cells were then removed and the cells were subcultured at 10 cells/well. Rat BMMNCs transduced with eGFP were expanded to 85PD. Alternatively, mouse MAPCs expanded to 80PD were used. Subcultures of undifferentiated MAPCs were generated by inoculating 100 MAPCs from cultures maintained for 75PD and further expanding them to greater than 5x ¹⁰⁶ cells. The expanded MAPCs were induced to differentiate into endothelium, neuroectoderm and endoderm in vitro. Lineage differentiation was confirmed by staining with antibodies specific to these cell types.

Example 5: Human MAPCs are not immunogenic

Mesenchymal stem cells have been shown to have low in vitro immunogenicity and to be able to engraft between allogeneic recipients (Di Nicola, M. et al. (2002) Blood 99:3838-3843; Jorgensen, C. et al. (2002) Gene Therapy 10:928-931; Le Blanc, K. et al. (2003) Scandinavian Journal of Immunology 57:11-20; McIntosh, K. et al. (2000) Graft 6:324-328; Tse, W. et al. (2003) Transplantation 75:389-397).

Figure 3 shows that human MAPCs exhibit low in vitro immunogenicity and are immunosuppressive when added to potent T cell MLRs (Tse, W. et al. (2003)). The results were consistent in all donor and responder pairs tested.

Responder and stimulator cells were prepared for these experiments, and MLRs were performed according to the procedures described by Tse, W. et al. (2003).

Example 6: MAPCs can regulate T cell responses

The ability of MAPCs to modulate (in this case, suppress) immune responsiveness is demonstrated by a T cell proliferation assay, which can be performed, for example, as follows.

Preparation of responder T cells

Responder cells are prepared from the lymph nodes of Lewis rats. Lymph nodes are surgically removed from rats and immediately placed in 3-5 ml culture medium (complete RPMI or complete DMEM) in a 60 x 15 mm plate. The lymph nodes are dispersed through a nylon filter (using the manual plunger of a syringe). The dispersion is placed in a 50 ml test tube and centrifuged at 1,250 rpm for 8 minutes. The supernatant (culture medium) obtained is discarded. The precipitate (containing cells) is resuspended in fresh culture medium (complete RPMI or complete DMEM). The cells are washed three times and then resuspended in fresh culture medium. The resuspended cell density is determined by counting the number of cells in a known volume. The cells are kept on ice. Before use, the cells are resuspended in culture medium (complete RPMI or complete DMEM) at a density of 1.25 x 10 ⁶ cells/ml.

Preparation of MAPCs

MAPCs were prepared from Lewis or Sprague-Dawley rats and then frozen as described above. They were thawed and then irradiated at 3000 rad. The irradiated cells were resuspended in culture medium (complete RPMI or complete DMEM) at a density of 0.4 x 10 ⁶ cells/ml, 0.8 x 10 ⁶ cells/ml, and 1.6 x 10 ⁶ cells/ml.

Preparation of Concanavalin A

Concanavalin A ("ConA") was used to activate T cells. ConA was dissolved in PBS (complete RPMI or complete DMEM) to a final concentration of 0 (PBS only), 10, 30, and 100 μg/ml.

Determination procedure

Each data point is based on at least three measurements.

20 μl of each ConA solution was added to each well of a 96-well, flat-bottomed microtiter plate, followed by 80 μl/well of responder cells and 100 μl/well of MAPCs. Each plate was incubated in a humidified incubator at 37°C, 5% _CO₂ for 4-5 days. During the final 14-18 hours of culture, each plate was pulsed with 1 μCi/well of ^3H -thymidine. Afterwards, the cells were automatically harvested on a glass fiber filter using a Tomtec harvester. Thymidine uptake was quantified in a microplate scintillation counter. Results are expressed as mean counts per minute (CPM) +/- SD.

The final concentration of ConA in the growth medium in each well was 0, 1, 3.16, and 10 μg/ml. The number of MAPCs present in each well was 0, 0.4, 0.8, and 1.6 x 10 ⁵ cells/well.

The results are shown in Figure 4 and discussed below.

result

Increasing amounts of ConA resulted in a dose-dependent stimulation of T cell proliferation (Figure 4, LN only). Lewis MAPCs inhibited the proliferation of ConA-stimulated T cells. This inhibition depended on the dose of MAPCs. Maximum inhibition (50%) occurred at the highest dose of MAPCs used in these experiments, when the ConA dose was low to moderate. These results are shown graphically in Figure 4 and indicate that MAPCs can inhibit the proliferation of activated T cells.

Example 7: MAPCs can inhibit the proliferation of stimulated T cells

The ability of MAPCs to suppress the proliferative responses of both syngeneic and mismatched (allogeneic) T cells was demonstrated using mixed lymphocyte reaction results. The following examples demonstrate the inhibitory effect of MAPCs on T cells from Lewis rats stimulated with irradiated splenocytes from DA rats. MAPCs from both syngeneic Lewis rats and mismatched (allogeneic) Sprague-Dawley rats inhibited T cell responses in a dose-dependent manner. The experiments were performed as follows.

Preparation of responder T cells

Responder cells were prepared from the lymph nodes of Lewis rats as described above.

Irradiated spleen stimulator cells

The spleen was surgically removed from DA rats. Splenocytes were then isolated from the spleen essentially as described above for the isolation of responder cells from Lewis rat lymph nodes. The isolated splenocytes were irradiated at 1800 rad. The cells were then resuspended at 4 x 10 ⁶ /ml and kept on ice until use.

Syngeneic MAPCs were prepared from Lewis rats as described above. Unmatched (allogeneic) MAPCs were prepared from Sprague-Dawley rats in the same manner. Both Lewis and Sprague-Dawley MAPCs were irradiated at 3000 rad and then resuspended in complete RPMI medium at densities of 0.03 x 10 ⁶ /ml, 0.06 x 10 ⁶ /ml, 0.125 x 10 ⁶ /ml, 0.25 x 10 ⁶ /ml, 0.5 x 10 ⁶ /ml, 1 x 10 ⁶ /ml, and 2 x 10 ⁶ /ml.

Determination procedure

Each well of a 96-well microtiter plate was loaded with: 100 μl of MAPCs (at each dilution described above) or 100 μl of culture medium; 50 μl of stimulator cell stock solution or control; 50 μl of responder cell stock solution or control; and culture medium (complete RPMI or complete DMEM) required to make the total volume equal to a final volume of 200 μl/well. All assays were performed using 96-well flat-bottom microtiter plates.

Each data point is based on at least three measurements.

The plates were incubated in a humidified incubator at 37°C, 5% _CO2 for 4-5 days. During the last 14-18 hours of culture, each plate was pulsed with 1 μCi/well of ^3H -thymidine. Afterwards, the cells were automatically harvested onto glass fiber filters using a Tomtec harvester. Thymidine uptake was quantified in a microplate scintillation counter. Results are expressed as mean counts per minute (CPM) +/- SD. Results

Exposure of T cells derived from Lewis rat lymph nodes (responder cells) to stimulator cells consisting of irradiated splenocytes from DA rats resulted in a very strong proliferative response of the responder cells, as shown in "No MAPC" in Figures 5A and 5B.

The addition of increasing doses of syngeneic Lewis MAPCs (Figure 5A) and non-matched (allogeneic) third-party Sprague-Dawley MAPCs (Figure 5B) resulted in significant and dose-dependent inhibition of T cell activation. The maximum inhibition level was approximately 80%. Even at the lowest dose of MAPCs, there was 40-50% inhibition.

As shown in Figures 5A and 5B, there was no ³ H-thymidine incorporation in the control, indicating that incorporation occurred solely due to the proliferation of activated responder T cells.

In summary, the results show that both syngeneic and third-party (allogeneic) MAPCs can suppress T cell proliferation even in the presence of potent activating splenocytes from unmatched rats. In these experiments, inhibition was highest when the number of stimulator cells, responder cells, and MAPCs in the reaction was similar (200,000 cells). Under these conditions, there was approximately 80% inhibition. Even when the ratio of MAPCs to other cells in the reaction was extremely low, there was significant inhibition. For example, there was 50% inhibition at 1.5% MAPCs (3,000 MAPCs compared to 200,000 of each other cell type). The results not only demonstrate that MAPCs have a strong immunosuppressive effect, but also that relatively small numbers of MAPCs are sufficient to suppress relatively large numbers of activated T cells even in the presence of very potent T cell activators.

Example 8: MAPCs are safe

The major immediate risk of intravenous injection of large numbers of cells is the accumulation of cell clumps in the lungs, which can lead to respiratory distress and possibly cardiac arrest. To confirm the safety of MAPCs in this regard, we measured the effect of intravenous injection of MAPCs on respiratory rate in Buffalo rats.

MAPCs were prepared from Lewis rats as described above ("Lewis MAPCs"). Splenocytes were also prepared from Lewis rats as described above for use as a control ("Lewis splenocytes").

One female Lewis rat per group served as spleen cell donor (experimental condition) and two Buffalo rats per group served as recipients (experimental condition).

Cells were administered to rats as follows. The data are presented graphically in Figure 6. The correspondence between each data point and the rat number is listed in the vertical legend to the right of Figure 6. All rats were Buffalo rats.

1.1, 1.210 x 10 ⁶ Lewis MAPC per mouse

2.1, 2.25 x 10 ⁶ Lewis MAPC per mouse

3.1, 3.22.5 x 10 ⁶ Lewis MAPC per mouse

4.1, 4.2, 1.2 x 10 ⁶ Lewis MAPC per mouse

5.1, 5.210 x 10 ⁶ Lewis spleen cells per mouse

6.1, 6.25 x 10 ⁶ Lewis spleen cells per mouse

7.1, 7.22.5 x 10 ⁶ Lewis spleen cells per mouse

8.1, 8.2, 1.2 x 10 ⁶ Lewis spleen cells per mouse

As described above, each rat was injected with 1.2, 2.5, 5 or 10*10 ⁶ MAPCs or 1.2, 2.5, 5 or 10*10 ⁶ splenocytes. This corresponds to 5, 10, 25 or 50*10 ⁶ cells/kg. Respiratory rate was measured before (0 minute) and 1, 5 and 10 minutes after intravenous injection of MAPCs or splenocytes. Respiratory rate was measured for 20 seconds, and then the count was multiplied by 3 to obtain respiratory rate per minute. Normal rat respiratory rate is between 60-140 breaths per minute.

result

All rats survived the intravenous cell injection. No significant differences or trends were observed in respiratory rate between conditions. The results are shown in Figure 6. Initial respiratory rate (0 minutes) was slightly lower because each rat was anesthetized before cell injection. At each time point, the measurements clustered together without any obvious trend.

In conclusion, intravenous injection of 5-50*10 ⁶ MAPCs/kg did not cause changes in respiratory rate or mortality in any rat under any conditions. The results confirm that intravenous injection of MAPCs is safe even at high doses.

Example 9: Expression of immune markers by MAPCs

The immunomodulatory properties of MAPCs were further characterized by measuring the expression of immunomodulatory markers in MAPCs, such as those described by Barry et al. (2005). These markers were measured using marker-specific antibodies and FACS analysis.

Rat bone marrow MAPCs were isolated, cultured, and harvested as described above. For FACS analysis, cells were suspended in PBS at 1-2 x ¹⁰ cells/ml. 200 μl of the cell suspension was added to each of a series of 12 x 75 polypropylene tubes. A marker-specific antibody or control was added to each tube, which was then incubated at room temperature for 15-20 minutes. At the end of the incubation period, 2 ml of PBS was added to each tube, followed by centrifugation at 400 x g for 5 minutes. The supernatant was discarded, and the cells in each tube were resuspended in 100 μl of PBS. Fluorescently labeled secondary antibodies were added to each tube in appropriate amounts, and the tubes were incubated again at room temperature for 15-20 minutes, this time in the dark. 2 ml of PBS was then added to each tube, and the tubes were again centrifuged at 400 x g for 5 minutes. The supernatant was discarded, and the cells in each tube were resuspended in 200 μl of PBS, which were then kept on ice pending FACS analysis.

The results are listed in Table 1. As shown in the table, rat MAPCs: (a) positive for class I MHC, CD11c, CD29, and CD90, (b) negative for class II MHC, CD11b, CD31, CD40, CD45, CD54, CD80, CD86, CD 95, and CD106. The negative results for each antibody were confirmed by positive staining of control cells (including rat peripheral blood cells and endothelial cells). These marker expression patterns (including those detected in MAPCs and those not detected) are completely consistent with the low immunoregulatory cross-section of MAPCs.

Table 1-Detection of immune cell-related markers in MAPCs

Example 10: MAPCs can suppress previously stimulated T cells in MLRs

cell

Responder cells were prepared from Lewis rat lymph nodes as described above. Splenocytes prepared from Buffalo or DA rats were used as stimulator cells as described above. MAPCs were prepared as described above.

program

MAPCs were added to the first set of MLRs simultaneously with the spleen cells (as in the previous examples). Additionally, MAPCs were added to a second set of MLRs with the same configuration as the first. However, the MAPCs in the second set were added three days after the spleen cells (or controls) were added. Thus, in the first set, MAPCs were added before the T cells began to proliferate in response to the spleen cells. In the second set, MAPCs were added three days into the T cell response to the spleen cells. All plates were incubated for a total of four days, and then pulsed with ³ H-thymidine and harvested as described in the previous examples. The experiment was otherwise conducted as described for the MLRs in the previous examples.

Each data point is based on at least three measurements.

result

As can be seen on the right side of Figure 7, MAPCs strongly inhibited T cell proliferation in MLRs when added three days after T cell stimulation with allogeneic splenocytes. Comparison of the left and right sides of Figure 7 demonstrates that MAPCs strongly inhibited the ongoing proliferative response. Quantitatively, the results show that for Buffalo cells, MAPCs inhibited T cell proliferation by 50% compared to controls when added three days after stimulation (right side of the figure), and by 75% when added simultaneously with stimulatory splenocytes (left side of the figure). Similarly, for DA cells, MAPCs inhibited T cell proliferation by 33% compared to controls when added three days after stimulation (right side of the figure), and by 70% when added simultaneously with stimulatory splenocytes (left side of the figure).

In all cases, the degree of immunosuppression by MAPCs generally depends on the number of MAPCs added to the reaction. In other words, immunosuppression depends on the dose of MAPCs added to the MLRs.

The publications cited above are herein incorporated by reference in their entirety with respect to the specific subject matter for which they are cited.

Example 11: MAPC inhibition of T lymphocytes is reversible

T cells were cultured with allogeneic stimulatory splenocytes in the presence or absence of irradiated MAPCs. After 13 days of culture, T cells were recovered and replated with irradiated allogeneic splenocytes in the presence or absence of MAPCs and cultured for 4 days.

T cell proliferation, which was suppressed in the presence of irradiated MAPCs during primary culture, was restored in the absence of MAPCs during secondary culture. The presence of MAPCs in secondary culture inhibited T cell proliferation comparable to that in primary culture, reaching a maximum of 90% inhibition at a MAPCs:T cell ratio of 1:2. The results, shown in Figure 8, clearly demonstrate that the inhibition of T cell proliferation by MAPCs is reversible in MLRs and that MAPCs can suppress both primary and secondary T cell proliferative responses.

Example 12: MAPCs can prevent GVHD

The ability of MAPCs to suppress GVHD was demonstrated in the following rat model. T cells from Buffalo rat donors served as a "graft" in Lewis x Buffalo rat recipients. The Buffalo transplanted cells were activated by the "foreign" Lewis cells in the host, leading to the development of GVHD in the model.

F1 recipient rats (Lewis x Buffalo) were sublethally irradiated with a single 600 rad dose on day 0. On the same day, they were injected intravenously with 2 x ¹⁰⁷ bone marrow cells and 10 x ¹⁰⁷ T cells (splenocytes) from donor Buffalo rats. Rats were monitored three times a week for signs of GVHD, which were assessed by posture, activity, fur texture, skin, and weight loss.

Group 1 did not receive MAPCs. Group 2 received 2.5 x 10 ⁶ MAPCs on day 1. Group 3 received 2.5 x 10 ⁶ MAPCs on days 1 and 8. There were five rats in each group. The results are shown in Figure 9. Administration of MAPCs significantly improved survival. 40% of the rats in Group 2 and 60% of the rats in Group 3 survived to the end of the experiment (42 days), while all rats that did not receive MAPCs (Group 1) developed GVHD by day 21 at the latest.

Claims (15)

1. Use of cells in the preparation of a medicament for adjuvant therapy to subjects at risk of or suffering from immune dysfunction, wherein the cells are pluripotent adult progenitor cells, characterized in that: the cells are not embryonic stem cells, embryonic germ cells or germ cells; are at least one cell type capable of differentiating into each of at least two lineages of endoderm, ectoderm and mesoderm embryonic lineages; express telomerase; have undergone at least 40 cell doublings in culture; are allogeneic or xenogeneic to the subject; do not induce a harmful immune response in the subject; and are administered via an effective route and in an effective amount as adjunct to one or more other treatments given to the subject. 2. The use of claim 1, wherein the cell is capable of differentiating into at least one cell type in each of the endoderm, ectoderm, and mesoderm embryonic lineages. 3. The use of claim 1, wherein the cells are positive for oct-3/4. 4. The use of claim 2, wherein the cells are positive for oct-3/4. 5. Use according to any one of claims 1 to 4, wherein the cell is allogeneic to the subject. 6. Use according to any one of claims 1 to 4, wherein the cells are administered to the subject at one or more doses comprising ^10⁴ to ^10⁸ cells per kilogram of subject mass. 7. The use of any one of claims 1 to 4, wherein the subject will receive or has received a graft and is at risk of developing host resistance to graft-versus-host disease or has already developed host resistance to graft-versus-host disease, and the cells are administered in conjunction with the graft. 8. The use of claim 7, wherein the graft is an organ, bone marrow, or blood graft, the subject is at risk of developing or has already developed a host-resistant graft reaction, and the cells are used in conjunction with the graft to treat the host-resistant graft reaction. 9. The use of claim 7, wherein the subject is receiving or will receive radiotherapy or chemotherapy or a combination of radiotherapy and chemotherapy, which weakens the subject's immune system, and the treatment with said cells is adjunctive to radiotherapy, chemotherapy or a combination of both. 10. The use of claim 7, wherein the cell is administered to a subject with an immune system deficiency. 11. The use of any one of claims 1 to 4, wherein the subject is at risk of or suffering from one or more of tumors, anemia or other blood disorders and immune dysfunctions, wherein the treatment with the cells is adjunctive to the treatment of these diseases. 12. The use of any one of claims 1 to 4, wherein the subject is at risk of or suffering from one or more of myeloproliferative disorders, myelodysplastic syndromes, leukemia, multiple myeloma, lymphoma, hemoglobinopathies, thalassemia, bone marrow failure syndrome, sickle cell anemia, aplastic anemia, Fanconi anemia, immune hemolytic anemia, congenital immunodeficiency, or autoimmune dysfunction, wherein the treatment with said cells is adjunctive to the treatment of these diseases. 13. Use according to any one of claims 1 to 4, wherein the cells are derived from bone marrow. 14. Use according to any one of claims 1 to 4, wherein the cells are derived from human bone marrow. 15. The use of any one of claims 1 to 4, wherein the subject is a human subject.