WO2005040346A2

WO2005040346A2 - Devices and methods for hematopoietic rescue or restoration

Info

Publication number: WO2005040346A2
Application number: PCT/US2004/035133
Authority: WO
Inventors: W. French Anderson; Yi Zhao
Original assignee: University Of Southern California
Priority date: 2003-10-24
Filing date: 2004-10-25
Publication date: 2005-05-06
Also published as: WO2005040346A3

Abstract

Methods and devices are also provided for rescuing or restoring the proliferation potential of hematopoietic cells. In particular, methods and devices are provided for rescuing or restoring a subject’s hematopoietic system following radiation therapy and/or chemotherapy without the need for a bone marrow transplant. Implantation of certain biological compositions (such as low-density bone marrow or lineage-negative cells) within a cell-impermeable pouch or sac, into an animal whose own hematopoietic ability has been severely compromised by radiation and/or chemotherapy treatment, for example, rescues or restores the hematopoietic capacity of the animal. The treatment can replace or augment bone marrow transplants, providing numerous advantages in the treatment of hematopoietic diseases, particularly for diseases where chemotherapy and/or radiation is administered, such as cancers generally, and more particularly blood-related disorders such as leukemias and lymphomas.

Description

DEVICES AND METHODS FOR HEMATOPOIETIC RESCUE OR RESTORATION

RELATED APPLICATIONS This application is related to and claims priority from U.S. Provisional application, Serial No. 60/421,371, filed on October 25, 2002, and U.S. Provisional application, Serial No. 60/347, 555, filed on October 26, 2001, which was expressly abandoned.

TECHNICAL FIELD This application pertains to devices and methods for rescuing or restoring the hematopoietic system of subjects, particularly humans, following radiation therapy and/or chemotherapy, and particularly for use in hematopoietic transplantations where stimulation, proliferation and/or expansion of blood cells is needed subsequent to radiation therapy and/or chemotherapy. BACKGROUND OF THE INVENTION The blood stem cell (hematopoietic stem cell) is of great interest in health and medicine. One particular application of blood stem cells arises in the treatment of blood cell disorders with bone marrow transplants. These disorders, including but not limited to leukemia, lymphoma, and myeloma, are cancers of the blood cell, characterized by production of excessive and non-functional blood cells. More specifically, leukemia is an acute or chronic disease of unknown cause in man and other warm blooded animals that involves the blood forming organs, is characterized by an abnormal increase in the number of leukocytes in the tissues of the body with or without a corresponding increase of those in the circulating blood and is classified according to the type of leukocyte most prominently involved. A lymphoma is a malignant tumor of the lymphoblasts derived from B lymphocytes. A myeloma is malignant tumor composed of plasma cells of the type normally found in bone marrow. One method of treating these disorders is to administer one or more doses of radiation therapy and/or chemotherapy. This procedure destroys the cancer cells, but also destroys the normal blood cells and/or other cells as well, and the patient is now unable to produce the blood cells necessary to sustain life. Further treatment is required, as otherwise the patient would die from the effects of the radiation therapy and/or chemotherapy, or suffer severe adverse health effects from the radiation and/or chemotherapy. A bone marrow transplant is then performed, which restores the ability of the patient to produce blood cells, thus rescuing the patient from the effects of the radiation therapy and/or chemotherapy. It is not precisely known which particular cells present in the donated marrow are responsible for this rescue, or which cell types are directly affected by the bone marrow transplant. Approximately 45,000 bone marrow transplants are performed worldwide each year, with about 20,000 procedures taking place in the United States. Allogenic bone marrow transplants usually require that the recipient take immunosuppressive drugs indefinitely to prevent rejection of the transplanted marrow, and the cost of the procedure averages approximately $200,000. Development of alternatives to bone marrow transplantation can provide significant medical benefits for the patient, as well as substantial cost savings. Immunosuppressive therapy is usually required for peripheral blood stem cell transplants as well. Exceptions to the requirement for immunosuppression occur when a genetically identical donor is available. This situation arises when the patient has an identical twin. This situation also arises when the patient is also the donor; that is, peripheral blood stem cells are isolated from the patient prior to a radiative ablation of the bone marrow.

Isolating hematopoietic stem cells from a leukemia or lymphoma patient for autologous donation eliminates the complications of transplant rejection and graft versus host disease. However, isolation of hematopoietic stem cells from a cancer patient also carries the risk that cancerous cells will contaminate the stem cell isolates, leading to re-introduction of the cancer cells along with the blood stem cells and defeating the entire purpose of the irradiation/chemotherapeutic procedure and transplant. Even if peripheral stem cell transplants alone are sufficient to rescue patients from potentially lethal irradiation, there are serious drawbacks to the procedure. Given the clinical difficulties and high costs of bone marrow transplants and stem cell transplants, there is clearly a need for therapies to rescue or restore a patient's blood sup ply, and that eliminate the need for bone marrow or stem cell transplants. Eliminating such transplants would also eliminate the need for immunosuppressive therapies to prevent transplant rejection and graft versus host disease. Finally, because suitable donors cannot be found for significant numbers of patients in need of bone marrow transplants, obviating the need for a bone marrow or stem cell transplant would provide life-saving therapy for people who have no alternative available.

SUMMARY OF THE INVENTION The invention comprises devices and methods for rescuing a subject that has been treated with a lethal or sublethal dose of chemotherapy and/or radiation, or a dose of chemotherapy or radiation which would significantly impair the health or hematopoietic ability of the subject without further intervention. In one embodiment, the invention comprises a method for rescuing a subject from a lethal dose of radiation or chemotherapy, or from a dose of chemotherapy or radiation which would significantly impair the health or hematopoietic ability of the subject without further intervention, by interacting the blood circulation of the subject with a biological composition. The biological composition is capable of stimulating hematopoietic stem cell proliferation or expansion. In one embodiment, the biological composition can be tissues or cells. In a preferred embodiment, the biological composition can be bone marrow, or can be derived from bone marrow. In another preferred embodiment, the biological composition comprises low density bone marrow, while in another embodiment the biological composition comprises lineage-negative cells. In certain embodiments, the biological composition comprising low density bone marrow or lineage-negative cells contains at least approximately 10,000,000 cells. In additional embodiments, the biological composition comprising low density bone marrow or lineage-negative cells contains approximately 10,000,000 cells. In certain embodiments, the biological composition is contained within an implantable sac. The implantable sac may be comprised of a membrane which is impermeable to cells, but permeable to biological macromolecules and biological molecules. In additional embodiments, the invention comprises a device for rescuing a subject from a lethal dose of radiation or chemotherapy, or from a dose of chemotherapy or radiation which would significantly impair the health or hematopoietic ability of the subject without further intervention. The device comprises an implantable sac impermeable to cells and permeable to biological macromolecules and biological molecules, as well as a biological composition contained within the implantable sac which stimulates hematopoietic cell proliferation or expansion when the device is implanted into a subject. The biological composition can comprise tissues or cells. In one embodiment, the biological composition is bone marrow, or is derived from bone marrow. In another embodiment, the biological composition comprises low density bone marrow, while in another embodiment the biological composition comprises lineage-negative cells. In certain embodiments, the biological composition comprising low density bone marrow or lineage- negative cells contains at least approximately 10,000,000 cells. In additional embodiments, the biological composition comprising low density bone marrow or lineage-negative cells contains approximately 10,000,000 cells. In additional embodiments, the invention comprises a method of rescuing a subject from a lethal or sublethal dose of radiation or chemotherapy. The method comprises the steps of implanting a cell-impermeable, biological macromolecule-permeable, biological molecule- permeable sac into a subject. The subject is then treated with a lethal or sublethal dose of radiation or chemotherapy, followed by addition of a biological composition to the interior of the device, which restores the hematopoietic capability of the subject. The biological composition can comprise tissues or cells. In one embodiment, the biological composition is bone marrow, or is derived from bone marrow. In another embodiment, the biological composition comprises low density bone marrow, while in another embodiment the biological composition comprises lineage-negative cells. In certain embodiments, the biological composition comprising low density bone marrow or lineage- negative cells contains at least approximately 10,000,000 cells. In additional embodiments, the biological composition comprising low density bone marrow or lineage-negative cells contains approximately 10,000,000 cells. In additional embodiments, after the device is implanted, a period of time from between 7 days to 10 days is allowed to pass before treating the subject with a lethal or sublethal dose of radiation or chemotherapy. In additional embodiments, the invention comprises a composition which contains at least one factor or at least one biological macromolecule or at least one biological molecule which is capable of rescuing a subject from a lethal or sublethal dose of radiation or chemotherapy. The composition can be isolated by interacting the blood circulation of an animal, which has been subjected to a lethal or sublethal dose of irradiation or chemotherapy, with a biological composition as detailed above, contained within an implantable sac.

DESCRIPTION OF THE DRAWINGS These and other features, aspects and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying figures where: Figure 1 is a FACS analysis of peripheral blood cells (mixture of cells from Ly5.1 and

Ly5.2 mice) with no antibody added; Figure 2 is a FACS analysis of peripheral blood from Ly5.1 mice stained with anti-I- 5.1 antibody; Figure 3 is a FACS analysis of peripheral blood from Ly5.2 mice stained with anti-I- 5.2 antibody; Figure 4 is a FACS analysis of peripheral blood from Ly5.1 mice stained with both anti-I-5.1 antibody and anti-I-5.2 antibody; Figure 5 is a FACS analysis of peripheral blood from Ly5.2 mice stained with both anti-I-5.1 antibody and anti-I-5.2 antibody; Figure 6 is a FACS analysis of peripheral blood from a Ly5.1 recipient mouse (mouse

1-1) implanted with a device containing low-density bone marrow cells from a Ly5.2 mouse donor; Figure 7 is a FACS analysis of peripheral blood from a Ly5.1 recipient mouse (mouse 1-2) implanted with a device containing low-density bone marrow cells from a Ly5.2 mouse donor; Figure 8 is a FACS analysis of peripheral blood from a Ly5.2 recipient mouse (mouse N-l) implanted with a device contaimng lineage-negative cells from a Ly5.1 mouse donor, 8 days after irradiation and loading of cells; Figure 9 is a FACS analysis of bone marrow from a Ly5.2 recipient mouse (mouse N-

1) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor, 8 days after irradiation and loading of cells; Figure 10 is a FACS analysis of spleen cells from a Ly5.2 recipient mouse (mouse N-l) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor, 8 days after irradiation and loading of cells; Figure 11 is a FACS analysis of cells removed from the device 8 days after irradiation and loading of cells from a Ly5.2 recipient mouse (mouse N-l) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor; Figure 12 is a FACS analysis of peripheral blood from a Ly5.2 recipient mouse (mouse N-2) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor, 14 days after irradiation and loading of cells; Figure 13 is a FACS analysis of bone marrow from a Ly5.2 recipient mouse (mouse N-

2) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor, 14 days after irradiation and loading of cells; Figure 14 is a FACS analysis of spleen cells from a Ly5.2 recipient mouse (mouse N-2) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor, 14 days after irradiation and loading of cells; and Figure 15 is a FACS analysis of cells removed from the device 14 days after irradiation and loading of cells from a Ly5.2 recipient mouse (mouse N-2) implanted with a device containing lineage-negative cells from a Ly5.1 mouse donor. Figure 16 shows Kaplan-Meier survival curves for various treatments, namely no device, the device without lin- cells, and the device plus lin- cells; the data show that the device with lin-cells can significantly improve animal survival to a lethal dose of radiation.

DETAILED DESCRIPTION

Overview The present invention provides devices and methods for stimulating one or more components of a patient's blood supp ly, for example, for stimulating blood stem cell proliferation, expansion, and/or differentiation. These methods and devices can replace or complement the use of certain procedures, such as bone marrow transplants and stem cell transplants whether these transplants be autologous or allogenic, and can serve as an adjuvant therapies to increase the success of certain procedures such as bone marrow transplants and stem cell transplants, radiation therapy and/or chemotherapy, and the use of hematopoietic stimulatory drugs, such as erythropoietin and/or granulocyte colony stimulating factor (G- CSF), or the like. The invention described herein comprises devices and methods for rescuing or restoring a subject's blood supp ly following radiation therapy and/or chemotherapy. The invention described herein also comprises devices and methods for rescuing or restoring hematopoietic ability to a subject which has a disease or condition resulting in a decrease or total loss of hematopoietic ability. Moreover, the invention can be used as a sole therapy or in conjunction with an additional therapy. . The invention described herein also comprises devices and methods for maintaining populations of blood cells, and for stimulating expansion, proliferation, and/or differentiation of blood cells. Embodiments of the invention include an implantable device which is filled with whole bone marrow cells, or one or more components of the bone marrow and/or the hematopoietic system, following radiation therapy and/or chemotherapy, where sufficient radiation and/or chemotherapy was administered to the subject to destroy cancerous or other pathological blood cells present in the hematopoietic system or in the bone marrow. Other embodiments of the invention include methods for using the device with a subject. Still other embodiments of the invention include placing one or more components of the hematopoietic system into the implantable sac that is implanted into the subject to replace or augment one or more non-functioning hematopoietic component(s) as needed by the subject. These latter embodiments may also be utilized with other therapies as required by the subject. The devices and methods of the present invention are employed to treat subjects suffering from cancers of the blood, other blood disorders, other types of cancers, and diseases requiring administration of radiation therapy and/or chemotherapy. Definitions As used in this disclosure, the following term are given the following meanings. By "subject" is meant an organism which is being treated using the devices and/or methods of the invention. Typically, the organism will be a mammal; preferably the organism is a primate; more preferably, the organism is a human. By "treat" is meant emp loying the devices and/or methods of the invention, with or without additional therapeutic agents and/or methods, in order to prevent or ameliorate either a disease or condition, or the symptoms of a disease or condition, or to retard the progression of a disease or condition or the symptoms of a disease or condition. Diseases and conditions include maladies iatrogenic diseases or conditions and include the side effects of therapeutic interventions, such as administration of doses of radiation therapy and/or chemotherapy. By "rescuing a subject' s hematopoietic potential, capacity or ability" after adrninistration of radiation therapy and/or chemotherapy is meant preventing a subject from dying due to the effects of the radiation or chemotherapy, or prolonging the life span of a subject for a longer period than would be expected after dose(s) of radiation therapy and/or chemotherapy, or increasing the quality of life of the subject. A subject treated by the devices and methods of the invention can survive an additional ten days, two weeks, one month, three months, six months, a year, three years, five years, or more than five years longer than would be expected were the subject not treated with the devices and methods of the invention. A "sublethal dose" of radiation or chemotherapy is defined herein as a dosage of radiation and/or chemotherapy which does not directly kill the subject to which it is administered, but which results in a significant impairment of the health of the subject, the subject's abil ity to function, or of the hematopoietic ability of the subject. "Rescuing" a subject fr om a sublethal dose of radiation or chemotherapy means either 1) that the recovery of the hematopoietic ability and/or blood system components occurs sooner than would otherwise be expected should the rescue not be performed, or occurs to a greater extent than would otherwise be expected should the rescue not be performed (recovery of the blood system components means that the number of cells or other products of hematopoiesis, as measured by the populations of various blood cells, including one or more than one of the various cell types that comprise blood cells, and/or of products of hematopoiesis such as platelets has reached a value that is within a range that is considered normal or healthy) or 2) the general health of the animal is improved, as measured by one or more than one clinical indicators, such as for example, susceptibility to infection, speed and effectiveness of clotting following cuts or injuries, body weight, appetite, endurance, or subjective measures of well- being. "Bone marrow" is defined as the soft, spongy tissue found in the center o f some large bones and that produces some of the components of blood. Bone marrow contains, among other substances, stem cells capable of repopulating the blood cells of the host from which it is taken. "Hematopoietic system" includes the bone marrow, and includes other tissues of the hematopoietic system, such as the spleen, thymus, liver and lymphatic system, where hematopoietic cells are formed, reside and/or can be mobilized into the circulation system of the subject. "Low density bone marrow" is defined as the fr action of bone marrow containing primarily mononuclear cells, but which can also contain other cells. "Lineage-negative cells" are de fined as that fraction of bone marrow cells which do not express a common lineage marker, often designated in the art as "lin^". " "Lineage-positive cells " are defined as that fraction of the bone marrow which express a common lineage marker. "Expansi on" in the context of hematopoietic stem cells refers to expanding the number of stem cells, in that a stem cell divides into two daughter cells, each of which is also a stem cell. "Proliferation " in the context of hematopoietic stem cells encompasses expansion of stem cells, as well as division of a stem cell into two daughter cells, one of which is a stem cell and one of which is a more differentiated cell, or of division of a stem cell into two daughter cells which are both more differentiated cells. Proposed Mechanism of Action The following discussion is presented for illumination of possible mechanisms by which certain embodiments of the invention operate. The invention is not limited, however, by the following proposed theories of operation. Lethal doses of radiation or chemotherapy are believed to cause death due to apoptosis, or programmed cell death, of damaged tissues. Damage to these tissues causes, among other deleterious effects, the loss of the hematopoietic ability of the subject to whom the radiation and/or chemotherapy has been administered. It is not clear whether hematopoietic ability is lost due to direct destruction of blood stem cells, or due to destruction of other tissues upon which the stem cells depend for survival. Bone marrow transplants rescue or restore the hematopoietic ability of a subject. It is not clear, however, which cellular or other components of the bone marrow are responsible for this effect. One possibility is that a secreted factor or factors, either present in bone marrow or secreted by cells present in bone marrow, stimulates blood stem cell survival, expansion, and proliferation. Another possibility is that direct cell-cell interactions are responsible for the survival, expansion and proliferation of the stem cells. Stem cell restoration may be due as a consequence of either the prevention of apoptosis of stem cells or the preservation of tissues on which the stem cells depend for survival. The research leading to the present invention demonstrates that a soluble factor or factors is likely to be sufficient for rescue or restoration of the hematopoietic ability of a patient following lethal or sublethal radiation and/or chemotherapy. Moreover, this phenomenon is applicable to other hematopoietic rescue or restoration events that are not necessitated by the effects of lethal or sublethal radiation and/or chemotherapy, such as where a subject has lost or diminished hematopoeitic ability. Description of Preferred Embodiments A membranous sac was implanted into recipient mice, with pore sizes which allowed exchange of biological molecules, nutrients, proteins, and other materials, but which prevented exchange of cells between the sac and the mouse into which the sac was implanted. After a period of time to allow for vascularization of the sac, the mice were lethally irradiated. A biological composition (e.g., low density bone marrow or lineage-negative cells) derived from a donor mouse was placed in the sac following the lethal irradiation. In the first group (group I; see Examples) two out of three mice survived the lethal irradiation and lived for approximately 100 days, at which point they were euthanized for further study. A control group of mice received the implanted sac, but the biological composition was not placed in the sac following lethal irradiation. These mice died within about two weeks of the lethal irradiation. The membranes of the implanted sac prevent cells from the donor biological composition from entering the recipient mouse and prevent cells from the recipient mouse from entering the interior of the sac. The rescue or restorative effect due to the implanted device containing the biological composition is thus most likely due to a factor or factors present in the biological composition that can diffuse through the membranes of the sac. This factor or factors is termed "Letha 1 Irradiation Rescue Factor," or LIRF for short (while LIRE is referred to in the singular, it may contain one or more active components). Also, while it is referred to as lethal irradiation rescue factor, it is also capable of rescuing a subject from lethal chemotherapy, and of rescuing a subject from sublethal doses of radiation and/or chemotherapy. By analogy with erythropoietin, a glycoprotein that stimulates production of red blood cells, and granulocyte colony stimulating factor, a protein that stimulates production of neutrophils, it is hypothesized that the factor or factors comprising LIRF are proteins or glycoproteins. LIRF may be present in the biological composition, or may be secreted by the cells in the biological composition in response to signals from the distressed recipient organism, or may be constitutively expressed by the cells in the biological composition. The invention also encompasses extracts and concentrates of cell media containing LIRF. The LIRF can be partially isolated by withdrawing a portion of the fluid from implanted sac at an appropriate time after the sac is loaded with low-density bone marrow, lin- cells, or other biological compositions containing one or more components of the hematopoietic system. Cells and cellular debris are removed from the fluid withdrawn, e.g. by centrifugation, and the remaining fluid can be utilized directly to rescue a subject from lethal or sublethal doses of radiation and/or chemotherapy, preferably by direct injection or by other routes of administration to the bloodstream of the subject. The fluid withdrawn can also be concentrated by methods known in the art, such as by evaporative methods or membrane filtration. LIRF may exert its effect by stimulating the expansion or proliferation of bone marrow hematopoietic stem cells and/or peripheral hematopoietic stem cells, and/or by preventing degradation of cells, tissues, and other elements in the recipient animal necessary for the expansion or proliferation of bone marrow blood stem cells and/or peripheral blood stem cells. Biological compositions suitable for use in the device and methods of the application include, but are not limited to, compositions comprising whole bone marrow, low density bone marrow, and lineage-negative cells. Typically, about 10,000,000 low-density bone marrow cells or 10,000,000 lineage-negative cells are introduced into the implantation device, however, greater or lesser amounts of bone marrow cells can be used, as will be understood by those with skill in the art with reference to this disclosure. Moreover, embodiments of the invention include the removal of a spent biological composition and replacement with a new biological composition comprising the same or different hematopoietic components. Implantation devices suitable for use in the application include any device which 1) allows cells, tissues, fluids, or any other biological material or biological composition from a donor animal to be implanted into the recipient animal; 2) segregates the implanted biological material or biological composition from the host animal, preventing the host immune system from attacking the implanted biological material or composition, and preventing cells in the implanted device from exiting the device into the host animal; and 3) allows exchange of nutrients, proteins, and other biological molecules between the circulation of the recipient animal and the biological composition in the implantation device. The implantation device should be capable of allowing one or more than one type of biological molecule or biological macromolecule selected from the group consisting of proteins, glycoproteins, phosphoproteins, peptides, lipids, fatty acids, monoglycerides, diglycerides, triglycerides, phosphoglycerides, sphingolipids, prostaglandins, steroids, carbohydrates, polysaccharides, nucleotides, oligonucleotides, vitamins, coenzymes, and nutrients required for cell survival such as oxygen or glucose, to exchange between the circulation of the recipient animal and the biological composition contained in the implanted device (in at least one direction, that is, either originating from the circulation of the recipient animal and crossing into the interior of the implanted device, or originating from the biological composition in the implanted device and crossing into the circulation of the recipient animal). In other embodiments, the device allows biological molecules and biological structures smaller than an organelle to cross between the implanted biological composition and the host animal, while preventing anything of the same size or larger than an organelle from traversing the device membrane. In other embodiments, the device has a molecular weight cutoff of about 1,000 kilodaltons, that is, the device permits biological macromolecules with a molecular weight of approximately 1,000 kilodaltons or less to traverse the device membrane, while preventing biological macromolecules and supramolecular assemblies with molecular weights above about 1,000 kilodaltons from exiting the device. Examples, of an implantation device useful in the methods of the present invention include the devices disclosed in United States Patent Nos. 5,314,417, 5,344,454, 5,421923, 5,453,278, 5,545,223, and 5,569,462, and in disclosed in Geller, R.L. et al., "Immunoisolation of tumor cells: generation of antitumor immunity through indirect presentation of antigen," Journal of Immunotherapy 20: 131-137 (1997), and include the TheraCyte^® device (TheraCyte, Inc., Irvine, California) that is an implantable chamber which can hold cells or other material. The TheraCyte^® system device has an outer membrane of polytetrafluoroethylene (PTFE), about 15 mm thick with pore sizes of 5 mm, designed to promote vascularization of the device, and an inner, cell impermeable membrane of PTFE with 0.4 mm pore size and 30 mm thickness, which prevents cells of the donor immune system from contacting the biological composition contained in the device chamber. Disorders and conditions that can be treated by the devices and methods of the invention include, but are not limited to, any diseases and conditions treated by radiation therapy and/or chemotherapy, particularly diseases and conditions where lethal or sublethal doses of radiation therapy and/or chemotherapy are administered. These diseases include, but are not limited to, neoplastic diseases, such as bladder cancer, brain cancer, breast cancer, colon cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, and cancers of the gastrointestinal tract, as well as other diseases and conditions characterized by abnormal cell growth or the abnormal lack of cell growth, proliferation or differentiation, such as congenital and acquired immune deficiencies, as will be understood by those with skill in the art with reference to this disclosure. Fluorescence-activated cell sorting (utilizing a flow cytometer), or FACS analysis, provides a means of rapidly measuring numerous physical and chemical characteristics of fluorescently labeled cells or particles as they travel in suspension, single file, past a laser light interrogation point. Any type of cells may be measured by this methodology as long as they are able to be placed in a single cell suspension. The FACS analysis is performed to determine the origin of cells in the recipient mice.

By using different mouse genotypes (for example, Ly5.1 mice as donors of the biological composition placed in the membranous sac, and Ly5.2 mice as recipients of the biological composition in the membranous sac), the origin of blood cells in the recipient's circulat ion can be determined. If implanted (recipient) mice have blood cells with no donor antigens, but only recipient antigens, then the reconstituted cells of the recipient mice did not originate from the donors, but rather from its own blood stem cells. FACS analysis utilizes a flow cytometer consisting of a light source, collection optics, electronics and a computer that translate light signals to data. The parameters measured by FACS analysis can be broken down into two parts: light scatter and fluorescence. Light scatter parameters can be used to measure physical characteristics such as cell size, granularity, membrane complexity and internal complexity. In the FACS analyses presented herein, the upper left hand corner plot presented in the Figures (the light scatter dot plot) represents the entire cell population or the "ungated" populat ion that is tested. This population can comprise lymphocytes, granulocytes, and macrophages. The "y" axis represents Forward Scatter (F S) and the "x" axis represe nts Side Scatter (SS). FS indicates the size of the cell and SS indicates the granularity of the cell. The x and y axes are in linear units. Note the smaller boxed-in area which represents a subgroup of the cells or the "gated" portion of the cells. Cells appearing outside of the smaller box in the left hand corner are dead cells and debris. The upper right hand corner plot presented in the Figures (the fluorescence dot plot) represents further analysis of the "gated" cells, broken down into cells which are detectable at certain wavelengths. Anti-a-5.1 monoclonal and anti-a-5.2 monoclonal antibodies are fluorescently labeled and are detectable at 575 nm and 525 nm respectively. The Gl box represents cells ("events") that can be detected at a wavelength of 575 nm, specifically cells which have Ly5.1 antigenic specificity but not Ly5.2 specificity. The G4 box represents cells that can be detected at a wavelength of 525 nm, specifically cells which have Ly5.2 specificity but not Ly5.1 specificity. The G3 box represents cells that are not recognized at either wavelength because they have no, or a very low concentration of, bound antibody, and the G2 box represents double positives, cells that have both anti-Ly5.1 and anti-Ly5.2 antibodies bound. The "y" axis and "x" axis represent fluorescence due to anti-Ly5.1 antibodies and anti-Ly5.2 antibodies, respectively, and are typically measured in log units. The present invention is further described by the following examples. While the experiments in the examples are generally performed in mice, the methods and devices of the invention can be employed to treat other mammals, more specifically primates, more specifically humans. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, are not to be construed to circumscribe or limit the scope of the disclosed invention. EXAMPLES Example 1: Preparation of Low-Density Bone Marrow Cells The Ly5.1 and Ly5.2 mice used in the experiments were C57BL/6J mice obtained from Jackson Laboratories (Bar Harbon, Maine, United States of America). Low-density bone marrow cells were isolated from C57BL/6J mice which had Ly5.2 antigenic specificity at the CD45 marker. A pooled total of approximately 5 x 10⁸ to 7 x 10^s whole bone marrow cells (approximately 100 ml) was collected from 10 mice. 25 ml portions of bone marrow were placed on top of 20 ml Nycomed Lymphoprepa™ buffer (Nycomed Pharma, AS, Oslo, Norway), for a sterile solution of 9.1 % w/v sodium diatrizoate and 5.7% w/v polysaccharide, a ready made sterile and endotoxin tested solution for the isolation of pure lymphocyte suspensions and bone marrow mononuclear cells). The tubes were centrifuged at 1800 rpm (800 g) in a Beckman GS-6R centrifuge for 20 minutes at room temperature. After centrifugation, three layers were apparent in the tube. The middle layer, containing the low- density bone marrow cells, was removed and used as the donor low density bone marrow cells for the following experiments. After pooling all portions obtained from processing the original approximately 5 x 10⁸ to 7 x 10⁸ whole bone marrow cells, approximately 1 x 10⁸ to 2 x 10⁸ cells were collected. Example 2: Treatment of Irradiated Mice with Low-Density Bone Marrow Cells Nine mice which have Ly5.1 antigenic specificity of the CD45 marker were divided into three groups of three. These mice are congenic with the Ly5.2 mice donors of the low density bone marrow cells, differing in genotype only for the CD45 marker. The first group of three, the Group I mice, had the TheraCyte^® system implantable bag placed under the skin of the back in accordance with the protocol recommended by the manufacturer. The mice were allowed to heal for 7 days, which allows time for vascular structures to form around the implanted bag. They were then exposed to a lethal amount of radiation of 1030 rads, in two doses of 515 rads per dose, with the second dose administered 3 hours after the first dose. After the second dose, approximately 10⁷ low density bone marrow cells from the congenic 5.2 mice donors were loaded into the device according to the protocol recommended by the manufacturer. The second group of three, the Group II mice, received a lethal amount of radiation of

1030 rads, in two doses, with the second dose administered 3 hours after the first dose. Immediately (within several minutes) after the irradiation, the TheraCyte^® system device, preloaded with approximately 10⁷ low density bone marrow cells from the congenic Ly5.2 mice donors, was implanted under the skin on the back of the mice, according to the protocol recommended by the manufacturer. The third group of three mice, the Group III mice, received a lethal amount of radiation of 1030 rads, in two doses of 515 rads per dose, with the second dose administered 3 hours after the first dose. They then simply underwent the procedure of having the skin on their back opened, and then closed. These mice served as the control group. Of the Group III mice, one mouse died on day 8 after irradiation, and the remaining two mice died on day 10 of irradiation. Of the Group II mice, one mouse died on day 6 after irradiation, one mouse died on day 8 after irradiation, and one mouse died on day 10 after irradiation. Of the Group I mice, one mouse died on day 21 after irradiation. The remaining mice (designated as mouse 1-1 and mouse 1-2) survived, and blood samples were taken at intervals. At the end of 100 days, the mice were euthanized and their spleens removed for study. Example 3: Origin of Restored Hematopoietic Cells In order to demonstrate that the cells which reconstituted the hematopoietic system of the irradiated animals originated from the recipient animal, and not the donor animals, fluorescence-activated cell sorting analysis (FACS analysis) was performed on cells from the peripheral blood of the recipient animals. At day 34 after irradiation, blood samples were obtained from the two surviving mice of Group I (designated mouse 1-1 and mouse 1-2). Labeled monoclonal antibodies (mAb's) against both the Ly5.1 and the Ly5.2 variants of the CD45 cell marker were purchased from Pharmagene. Since the donor mice have the Ly5.2 isotype and the recipient mice have the Ly5.1 isotype, reconstitution of the hematopoietic system of the recipient animal by cells from the donor animal will result in the presence of blood cells having the Ly5.2 marker in the recipient animal. However, if the hematopoietic system of the recipient animal is reconstituted from its own stem cells, its resulting blood cells will bear the Ly5.1 marker. The analyses are shown in Figures 1-15. Figures 1-15 all represent fluorescence- activated cell sorting analysis (FACS analysis) data. Fluorescence from the anti-5.2 mAb is plotted along the x-axis, while anti-5.1 mAb fluorescence is plotted along the y-axis Figure 1 is a control experiment where no antibody is added to the cells used in the FACS analysis; no fluorescence signal is seen in the Gl or G4 regions of the upper right hand plot. A mixture of approximately equal portions of cells from Ly5.1 mice and Ly5.2 mice was used in Figure 1. Figure 2 is a control experiment where 5.1 cells are stained with anti-5.1 mAb; significant fluorescence is seen in the Gl region of the upper right hand plot. Another, much smaller population of cells is observed in region G3 at a much lower level of fluorescence; this is due to contamination by a small number of non-blood cells with CD45 markers. Figure 3 is a control experiment where pure 5.2 cells are labeled with anti-5.2 mAb; a high level of fluorescence along the x-axis (region G4 of the upper right hand corner plot) is observed for the population of 5.2 cells. Again, a much smaller population of cells is observed at a much lower level of fluorescence due to contamination by a small number of non-blood cells with CD45 markers. Figure 4 is another control, where pure 5.1 cells are mixed with both anti-5.1 mAb and anti-5.2 mAb. A similar pattern is seen as that in Figure 2; however, another small population is seen showing high fluorescence for both anti-5.1 mAb and anti-5.2 mAb. This is due to a low amount of cross-reactivity of the anti-5.2 mAb with the 5.1 cells. If 5.2 cells were actually present, they would appear in region G4, and thus the true presence of 5.2 cells can easily be distinguished from the artifacts caused by mAb cross-reactivity. Figure 5 is a control, where pure 5.2 cells are mixed with both anti-5.1 mAb and anti-5.2 mAb. The pattern of reactivity is similar to that of Figure 3. Figures 6 and 7, from mouse 1-1 and mouse 1-2, respectively, demonstrate that the reconstituted blood cells come from the rescue of the recipient mouses' hematopoietic cells, and not from leakage or other transfer of the donor cells in the implanted device. In Figure 6, a strong signal is seen along the y-axis in region Gl of the upper right hand plot, indicating a high population of cells with the 5.1 marker; a weaker signal is seen in region G2, where anti- 5.2 mAb cross-reacting with 5.1 cells is detected; no signal is seen in region G4, where fluorescence due to 5.2 cells would be expected. A similar pattern is seen in Figure 7. Thus the cells detected in the recipient animals, bearing Ly5.1 markers, originated from the recipient Ly5.1 animals and not from the donor Ly5.2 animals. Example 4: Isolation of Lineage-Negative Cells Bone marrow cells were harvested from the femurs and tibias of Ly5.1 mice. After lysis of red blood cells with ammonium chloride lysis buffer (Ortho-munea Lysing Reagent (Ortho Diagnostic Systems, Inc., Raritan, NJ US) describing an ammonium chloride lysis buffer), cells were stained with biotinylated antibodies to lineage markers. Lin⁺ cells were depleted with streptavidin-conjugated magnetic beads by using a CS column (Miltenyi Biotech, Auburn, CA US). The lineage depleted cells were collected and used in the following experiments. Example 5: Treatment of Irradiated Mice with Lineage-Negative Cells A time course was conducted to determine whether the time from implanting the device to loading the cell matters for the radiation rescue. Eleven mice were implanted with the TheraCyte^® system device. Two mice received a

1030 rad dosage of irradiation (as in Example 1) at 8 days after implantation; two mice received a 1030 rad dosage of irradiation in two doses of 515 rads each (as in Example 1) at 10 days after implantation; two mice received a 1030 rad dosage of irradiation (as in Example 1) at 12 days after implantation; three mice received a 1030 rad dosage of irradiation (as in Example 1) at 14 days after implantation; and two mice received a 1030 rad dosage of irradiation (as in Example 1) at 16 days after implantation. Immediately after irradiation, the implanted devices were each loaded with approximately 1 x 10⁷ lineage-negative cells, isolated as in Example 4. It should be noted that the recipient mice in these experiments are Ly5.2 mice and the donor mice are Ly5.1 mice, the reverse of the recipient/donor arrangement in Examples 1-3. Of these mice, only the two mice which were irradiated at 8 days post-implantation survived. These mice are designated as mouse IV-1 and IN-2. A second set of two mice were then implanted with the empty device and irradiated at eight days post-implantation, followed by loading of the device with approximately 10⁷ lineage- negative cells. These mice are designated as mouse N-l and N-2 (as with mice IN-1 and JN-2, the recipient mice N-l and V-2 are Ly5.2 mice and the donor mice from which the Lin- cells are isolated are Ly5.1 mice). Mice IN-1 and IN-2, and mice N-l and N-2, showed no donor cell contribution in the reconstitution of their blood cells. Experiments demonstrating this conclusion are detailed in the next examples. Example 6: Determination of Donor Cell Contribution in Hematopoietic Reconstitution Mice N-l and N-2 were sacrificed at day 8 and 14, respectively, after cell loading to determine the origin of reconstitution of blood cells in the recipient. There was no donor cell contribution in blood, bone marrow, and spleen. In other words, all the reconstituted cells were derived from endogenous cells in the recipients. Figures 8, 9, and 10 are FACS analyses of the recipient mouse N-l peripheral blood cells, bone marrow, and spleen, indicating that reconstituted cells were of recipient origin. Figures 12, 13, and 14 are FACS analyses of the recipient mouse N-2 peripheral blood cells, bone marrow, and spleen, again indicating that reconstituted cells were of recipient origin. The cell origin in the devices at day 8 and 14 after cell loading was also checked and there were no cells from the recipient mice in the device. Figure 11 is an analysis of the cells in the device implanted into mouse N-l, euthanized at 8 days post-irradiation; Figure 15 is an analysis of the cells in the device implanted into mouse N-2, euthanized at 14 days post- irradiation. This observation proves that the device is effectively preventing any cell-cell exchange through the device membrane, strongly implying that the rescue effect observed results from a systematic, soluble factor or factors which can diffuse through the device membrane. Example 7: Bone Marrow Cellularity in Mice with and without Device The tables below summarize the results of experiments to determine bone marrow cellularity of mice that have received a lethal dose of radiation which either have or have not been treated with the methods and devices of the application. Mice without the device are represented as the control. Mice with the device are represented as such.. Table 1 represents the raw control data. Table 2 represents the raw device data. Table 3 represents the comparison of the mean of the control and the mean of the device data. The data in Table 3 indicates that the number of bone marrow cells in mice with the device are significantly increased over the level of cells in control mice. Table 1 Control Day 0 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 58000000 1100000 460000 220000 240000 230000 100000 55000000 830000 540000 230000 260000 130000 300000

Average 56500000 965000 500000 225000 250000 180000 200000 SD 7071.0678 : 14142.136 70710.678 141421.4

Table 2 Device Day 7 Day 8 Day 10 Day 14 1400000 1400000 530000 820000 750000 700000 400000 520000 760000 430000 810000 1000000 520000 1400000 700000 250000 480000 Average 1062000 1050000 472857.14 716666.7 SD 324376.32 494974.75 137805.94 170391.7

Table 3 Day 0 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Control 56500000 965000 500000 225000 250000 180000 200000 Device 1062000 1050000 472857.14 716666.7 Increase 4.72 4.2 2.6269841 3.583333

Example 8: Radiovrotective Effects of Donor Cells ^■ in Device An immunoisolate device (ID) (Theracyte^®) was used to test if soluble factors produced from bone marrow cells could rescue lethally irradiated animals. The IDs were implanted subcutaneously on the back of mice. At different time points post implantation (3, 5, 7, 8, 10, 12, 14, 16 day post implantation), mice received a lethal dose of radiation. About 1 hour post radiation, 1 x 10⁷ lin- cells or low-density cells were loaded into the IDs. One group of animals received ID implantation and cell loading immediately after lethal radiation. Animal survival was monitored daily. As shown in figure 16, when radiation and cell-loading were performed 7 to 8 days post ID implantation, 78% (28 out of 36 mice) animals survived, while 11% (2 out of 18 mice) animals survived with ID implantation and PBS infusion only. There was 3% (1 out of 33 mice) animal survived in the control group (radiation only, no ID implantation). We tested the time course of the rescue event, and the data is shown in Table 4. There is no rescue when ID/cell-loading is administered immediately after the radiation, or at any other time point tested, such as 12, 14 and 16 days post ID implantation. Moreover, less effective rescue is observed when cell-loading is performed 10 days after the TID implantation. These phenomena suggest that the lin-cell rescue can only occur in a very narrow period of time. Table 4. Implanted ID /lin- cell mediated lethal radiation rescue effect in a narrow window time.

Beside lin- and low density cells from bone marrow, we also tested several cell types in ID/cell loading system. However, none of the tested cell types (3T3, stromal cells, CHO, 3T3- Ll, endothelia cells) produced a rescue effect, suggesting that the rescue event is a specific effect found only with a bone marrow cell subset. Interestingly, whole bone marrow cells also did not give rescue, this may indicate that the cells play the rescue function is enriched in lin- population. Example9 : Secondary Bone MarrowTransplantation Experiment To determine if the long-term repopulating hematopoietic cell compartment is rescued, secondary bone marrow transplantation experiments were performed. Bone marrow cells from surviving mice were isolated at different times after lethal dose radiation and transplanted to second lethally irradiated mice to observe the animal survival rate and donor cell reconstitution in peripheral blood. The donor cells (2 X 10⁶) weie obtained by mixing 2 to 3 rescued mice bone marrow and injected via tail vein immediately after lethal dose radiation of the recipient mice. As shown in Table 5, in 4 individual experiments of total 16 recipient mice, all of the animals survived at least 4 months (experiment 4) and some more than 12 months. On average, at least 50% of peripheral blood cells were originated from donor cells when examined 4 weeks post BMT. It is noteworthy that we did observe high long-term reconstitution of the blood system, as shown in experiment 2 and 3.

Table 6: Summary of BMT with rescued bone marrow cells

The various publications, references, patents, and patent applications mentioned herein are hereby incorporated by reference herein in their entireties. Although the present invention has been discussed in considerable detail with reference to certain preferred embodiments, other embodiments are possible. Therefore, the scope of the appended claims should not be limited to the description of preferred embodiments contained in this disclosure. All references cited herein are incorporated by reference to their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for rescuing or restoring hematopoietic ability in a subject in need thereof, comprising: interacting the blood circulation of the subject with a biological composition comprising one or more hematopoietic components that modulate hematopoietic cell production.

2. The method of claim 1, wherein the biological composition is derived from bone marrow.

3. The method of claim 1, wherein the biological composition comprises low density bone marrow.

4. The method of claim 3, wherein the low density bone marrow contains at least approximately 100,000 cells.

5. The method of claim 1, wherein the biological composition comprises lineage-negative bone marrow cells.

6. The method of claim 1, wherein the biological composition is contained within an implantable sac.

7. The method of claim 6, wherein the implantable sac comprises a membrane impermeable to cells.

8. The method of claim 7, wherein the membrane of the implantable sac is permeable to biological macromolecules.

9. The method of claim 1, wherein one or more component(s) of the patient's blood supply is depleted following radiation therapy and/or chemotherapy.

10. A device for modulating the blood supply of a subject comprising: an implantable sac impermeable to cells and permeable to biological macromolecules; and a sample of a biological composition, comprising one or more hematopoietic components contained within the implantable sac, wherein the one or more hematopoietic components modulate hematopoietic cell expansion, proliferation and/or differentiation when the device is implanted into a subject.

11. The device of claim 10, wherein the device is placed into the subject prior to or following lethal or sublethal radiation and/or chemotherapy.

12. The device of claim 10, wherein the sample of the biological composition is removed and replaced with another sample of the same or a different biological composition.

13. The device of claim 10, wherein the biological composition is derived from bone marrow.

14. The device of claim 10, wherein the biological composition comprises low density bone marrow.

15. The device of claim 10, wherein the biological composition comprises lineage-negative bone marrow cells.

16. A method of rescuing or restoring the blood supply of a subject comprising: implanting the device of claim 10 into the subject in a manner such that additional compositions can be added to the interior of the device at a later time; treating the subject with radiation and/or chemotherapy; and adding a biological composition to the interior of the device which restores the hematopoietic potential of the subject.

17. The method of claim 16 wherein the biological composition comprises tissues or cells.

18. The method of claim 16, wherein after the device is implanted, a period of time from approximately between 7 days to 10 days is allowed to pass before treating the subject with radiation therapy or chemotherapy.

19. The method of claim 16, wherein the biological composition is derived from bone marrow.

20. The method of claim 16, wherein the biological composition is low density bone marrow.

21. The method of claim 16, wherein the biological composition comprises lineage-negative bone marrow cells.

22. A composition comprising at least one soluble factor that rescues or restores the blood supply of a subject following lethal or sublethal radiation and/or chemotherapy.

23. An allogenic hematopoietic rescue device comprising: an implantable sac impermeable to cells and permeable to biological macromolecules; and a sample of a biological composition, comprising one or more allogenic hematopoietic components contained within the implantable sac that modulate hematopoietic cell expansion, proliferation and/or differentiation when the device is implanted into a subject.

24. The allogenic hematopoietic rescue device of claim 23, wherein the allogenic hematopoietic component includes hematopoietic progenitor and/or stem cells.