DIABETES CELL THERAPY

[0001] This application claims priority to U.S. Provisional Application No.

61/200438 filed November 28, 2008 and is expressly incorporated by reference.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

REFERENCE TO MICROFICHE APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] This invention relates to the treatment of diabetes using stem cells.

BACKGROUND OF THE INVENTION

[0005] Diabetes is a global and growing epidemic. In the United States alone, 23.6 million children and adults (7.8% of the population) have diabetes. Current treatment for Type 1 Diabetes does not provide a cure and is very disruptive to quality of life. It also creates massive costs and expenditures for both the individual and our healthcare systems. In the U.S., the total annual economic cost of diabetes in 2007 was an estimated $174 billion. Other autoimmune disorders, including rheumatoid arthritis, Charcot-Marie-Tooth disease, Crohn's disease, Addison's disease, Graves' disease, lupus erythematosus, myasthenia gravis, pernicious anemia, and multiple sclerosis also pose substantial burdens to our health and health care system.

[0006] Building on the successful use of bone marrow-derived stem cells for the treatment of blood cancers, many kinds of stem cell therapies are being investigated to help treat diseases for which there is no current cure.

[0007] Cell therapy describes the process of treating a medical condition by replacing diseased or dysfunctional cells with healthy, functioning ones. A classic example is bone marrow therapy for cancer, whereby the patient is irradiated and the bone marrow repopulated with healthy bone marrow. In addition to bone marrow cells, a large range of cells can serve in cell therapy including blood cells and mature and immature solid tissue cells.

[0008] Stem cell therapy is a more recent type of cell therapy specifically using stem cells to treat a medical condition. Stem cells are primitive, unspecialized cells that have the capacity to self-renew and to differentiate into mature, specialized cells. Depending on their origin, human stem cell preparations are either embryonic stem cells — derived from embryos — or adult stem cells — derived from various adult tissues.

[0009] Human embryonic stem cells were first derived from human blastocysts in 1998. They are totipotent, meaning they can become any of the more than 200 known differentiated cell types of the human body. Embryonic stem cells are found in the embryo until about five days after fertilization.

[0010] Adult stem cells — also known as somatic stem cells — exist in many tissues of the human body (in vivo) at any age after birth (newborn to adult), and are the body's own mechanism for tissue turnover and regeneration to repair specific damage or normal 'wear and tear.' Adult stem cells in the human body are quite rare compared to the somatic cells and often are difficult to identify, isolate and purify. They are most commonly identified by protein markers on their surface, such as CD34 on hematopoietic stem cells (HSCs), one category of adult stem cells.

[0011] Induced pluripotent stem cells (iPSC) are pluripotent stem cells derived from somatic cells by treatment with specific proteins, nucleic acids, and/or viruses.

[0012] Proven and potential clinical benefits underlie the strong interest in stem cells and stem cell therapy. In 1968, bone marrow was first used to source HSCs for transplantation to regenerate a patient's blood and immune system after myeloablative conditioning. Currently there have been successful adult stem cell therapy cases in more than 90 medical indications ranging from cancers to myocardial infarction (after a heart attack) using bone marrow and peripheral blood (blood that circulates in the body) as the source for HSCs, and several MSC products are in clinical trials. Additionally, adult stem cells are being utilized currently to enhance other proven medical methods of addressing blood and tissue cancer, disease and degeneration, such as chemotherapy, pharmaceutical drugs, organ transplants and orthopedic scaffolds. At least 300 million people in the U.S., E.U. and Japan have medical conditions that could potentially benefit from known stem cell therapeutics currently in development.

[0013] Adult stem cells can be broadly subdivided into pluripotent hematopoietic stem cells (HSC) and multipotent mesenchymal stromal cells (MSC). HSCs regenerate all cell types in the blood and the immune system, while MSCs can regenerate many tissues including bone, fat, cartilage and muscle. The bone marrow contains both HSCs and MSCs. To date, adult stem cells have been detected in many tissues including: bone marrow, blood (umbilical cord and peripheral blood), brain, dental pulp, cornea, liver, skin, adipose tissue, and heart.

[0014] Non-hematopoietic bone marrow stromal cells (MSC) within adult bone marrow, including reticular cells, smooth muscle cells, adipocytes and osteoblasts, provide the local environmental cues necessary to support the survival, proliferation and differentiation of hematopoietic stem cells (HSC). Alexander Friedenstein and associates were the first to demonstrate in the '70s that bone marrow explants placed in the kidney of recipient mice could form bone-like tissue that was capable of self-renewal and self- maintenance and could support the formation of blood cells. He further described the isolation from the bone marrow of spindle-shaped cells that grew adhered to plastic in tissue cultures, which he defined as colony- forming unit fibroblasts (CFU-Fs), that were able to generate fat, bone and cartilage in vitro. Friedenstein went on to show that CFU-F- derived stromal cells served as feeder layers for the culture of blood forming cells in the marrow, the HSC.

[0015] Subsequent studies have supported the hypothesis that stromal populations are derived from multipotential bone marrow stromal cells (BMSC) or subsets that are also referred to as bone marrow stromal stem cells (BMSSC), mesenchymal stem cells/marrow stromal cells (MSC), marrow-isolated adult multipotent inducible cells (MIAMI), multipotent adult progenitor cells (MAPC) and mesenchymal adult stem cells (MASCS).

[0016] MSC research has expanded greatly since the pioneering work of

Owen and Friedenstein in the last two decades. Originally termed CFU-F (colony forming unit fibroblast) and described based on their adherent properties, MSCs are now described using a phenotype based on several markers including CD73 and CDl 05 and lack of CD45 expression. The general consensus for human MSC used in several current clinical trials appears to be that of fibroblastic cells from bone marrow with a phenotype of CD73+, CD90+, CD105+, CD45-.

[0017] The immune phenotype of MSCs (widely described as MHC

I+, MHC H-, CD40-, CD80, CD86-) is regarded as nonimmunogenic and, therefore, transplantation into an allogeneic host may not require immunosuppression. MHC class I may activate T cells, but, with the absence of costimulatory molecules, a secondary signal would not engage, leaving the T cells anergic. Many reports have also described MSCs as having immunosuppressive properties — specifically that MSCs can modulate many T-cell functions including cell activation. This suppression appears to be independent of MHC matching between the MSCs and the T cells. Some reports have demonstrated that direct cell-cell contact is required for suppression, whereas others have shown that the suppressor activity depends on a soluble factor. It has also been shown that MSCs have immunomodulatory properties impairing maturation and function of dendritic cells and that human MSCs inhibit in vitro human B-cell proliferation, differentiation, and chemotaxis.

[0018] MSCs show great promise as a biological therapeutic for a diverse range of unmet medical needs. The reasons for this are many and include: ease of isolation and expansion in culture, multipotency, paracrine effects, immunomodulatory properties, migratory behavior and favorable ethical considerations. In recent years it has also come to light that MSC plasticity extends beyond the conventional bone, adipose, cartilage, and other skeletal structures, and has expanded to the differentiation of liver, kidney, muscle, skin, neural, and cardiac cell lineages. Thus, their use is expected to further increase as a variety of disease conditions can be treated with these cells.

[0019] Previous attempts at stem cell therapy of type I diabetes have been made, but each immuno-ablated or immunosupressed the patient and used purified and/or amplified stem cells for the transplant. Each of these techniques has obvious disadvantages. Immunoablation and immunosupression leave the patient significantly immunocompromised and at risk for both infections and cancer. Further, the natural environment is greatly perturbed, which can lead to unknown consequences. Purifying cells means that very few are available for use and those few cells are removed from their normal supportive environment, providing opportunity for the cells to change. Amplification of the cells to increase their number means that the cells are both removed from a normal environment and forced to reproduce in an abnormal environment. Thus, the transplanted cells will probably have changed in unanticipated ways due to their treatment, and this may lead to less efficacy and unintended consequences.

[0020] What is needed in the art is an effective method of treating patients with cell therapy that lacks these drawbacks.

SUMMARY OF THE INVENTION

[0021] The following abbreviations and/or definitions are used herein:

[0022] In one embodiment, the invention generally relates to method of treating a patient with whole bone marrow or marrow stem cells, wherein the cells are harvested following low dose mobilization of MSCs, as defined below. Such cells can be used to treat any disease currently treated with bone marrow or marrow derived stem cells. The cells can be used as is, or can be further purified or amplified before use pursuant to existing treatment protocols, and if needed the treatment can be combined with other treatments such as immunosupression and the like. The method can be used to treat any disease that is responsive to bone marrow therapy, including those described in Table 1 and can be combined with most, if not all of the existing methodologies, including those of Table 2.

[0023] "Low dose mobilization" is defined herein as that low dosage over a period of 3-9 days of a mobilization factor, sufficient to raise circulating MSC level, as assessed by CD34+ cell counting, to 0.02-0.05%. hi a preferred embodiment, target CD34+ levels are 0.03%, corresponding to a CD34+ count in the bone marrow of about 0.12X10 ⁸ cells/kg body weight. If after 5 days the CD34+ levels do not reach 0.03%, the patient will receive 2 additional days of mobilization, and CD34+ cells will be subsequently re-measured. If the target of 0.03% has been reached, the patient continues with the procedure, otherwise the patient is excluded from the protocol. Failure to achieve mobilization in 7-9 days indicates that the dose is too low and should be increased.

[0024] All numerical values provided herein are provided within the experimental accuracy of the measuring instrument employed. Thus, if flow cytometry has a +/- 20% error rate, a 0.03% level ranges from .024-0.036.

[0025] CD34+ cells found in the peripheral circulation are measured by flow cytometry of cells labelled with fluorescently labeled anti-CD34 antibodies, and low dose mobilization continues until the requisite level is achieved, as described above.

[0026] As used herein, "mobilization factor" means a pharmaceutically acceptable agent that stimulates the physiological increase of stem and progenitor cells in the bone marrow, and includes among others G-CSF, IL-8, Mobozil, POL6326, Flt3L, PTH and cyclophosphamide and active variants and combinations thereof and other treatments having similar effects. [0027] In a preferred embodiment, mobilization is achieved by a dosage of

3-15 μg/kg of G-CSF, as twice-daily or once-daily injections. In a more preferred embodiment, mobilization is achieved as once-daily injections of 5μg/kg IV Filgrastim.

[0028] In another embodiment, the invention relates to method of treating type I diabetes patients with untreated bone marrow cells, wherein the patient is not pretreated by immunoablation or immunosupression, and the therapeutic cells are obtained directly from the bone marrow (without intervening purification and/or amplification) following low dose mobilization of MSCs.

[0029] The phrase "untreated bone marrow" or "whole bone marrow" means bone marrow cells that are not purified for one or more cell types, but rather comprise the normal mobilized marrow cell population, and are not amplified after harvesting in any way. Specifically included within the scope of these phrases are minor treatments such as filtration or centrifugation to remove bone fragments and blood clots, and the addition of other agents such as clotting agents or antibiotics and other drugs.

[0030] The phrase "the patient is not immunoablated or immunosuppressed" means that the patient is not or has not been recently treated with radiation or chemical agents to suppress or destroy the bone marrow or immune system. Specifically excluded from the term is the endogenous levels of immunosupression that may arise from lifestyle choices or disease, as well as any immunosupression that may arise from subsequent transplant of stem cells, bone marrow, or MSCs.

[0031] The term "patient" includes human and animal patients.

[0032] In some embodiments of the invention autologous cells are preferably transplanted, but in others allogeneic transplants are preferred or a mixture thereof. In another embodiments, cell are administered at the same time as a factor to enhance targeting of the stem and progenitor cells to the organ and increasing organ function. Targeting factors include, without limitation, DPPIV, TNFa, VEGF, IGF, EGF, gastrin, fucosyl transferase, and aldehyde dehydrogenase inhibitors such as DEAB, as well as other treatments achieving similar effects.

[0033] In another embodiment, the transplanted cells are a mixture of autologous bone marrow stromal cells and allogeneic mesenchymal stem and progenitor cells derived from donor stromal tissues including the umbilical cord matrix, Wharton's jelly, perivascular cells surrounding the umbilical cord vessels, placental cells including amniotic, chorionic, epithelial and endothelial progenitors, cord blood derived mesenchymal progenitors including unrestricted somatic stem cells and endothelial progenitors, menses-derived cells with pluri-lineage potential and generally cells that are positive for one or more of CDl 05, CD90, CD73, CD44, Stro-1, VCAM but negative for CD45, CD34, HLA-DR, and bone marrow stromal cells derived from bone marrow puncture.

[0034] In another embodiment the allogeneic cells are derived from a donor of fetal islet cells combined with autologous bone marrow derived cells, hi a more preferred embodiment, the cells are a mixture in the range of 3:1-2:1 autologous to allogeneic cells ranging from 1-10 ⁶ cells/kg. In an even more preferred embodiment, the cells comprise morphologically small, rapidly self-renewing cells from the bone marrow stroma.

[0035] In another embodiment, the cells are autologous stem cells induced toward the formation of islets, mixed with alginate or other polymer or matrix, or with other donor cells, or targeting agents, or antibodies.

[0036] The invention also relates to a method of delivering cells for cell therapies, wherein the cells are injected into an artery at or near the target location, whereby the proximity to the target site facilitates mobilization of the cells into the target tissue, and the delivery method causes little or no trauma to that tissue. For Type I diabetes, preferably the bone marrow cells are injected directly into a pancreatic artery, and most preferably, the cells are placed into one or both of the superior mesenteric or celiac trunk artery.

[0037] Another embodiment of invention is a novel catheter that can be used to deliver cells or other therapeutics directly to pancreatic internal arteries with less risk of damage to the artery. Figure 1 depicts a modified catheter designed to release the cell sample into the pancreatic artery. There is a need for such a catheter because all of the existing commercially available abdominal catheters require that catheterization occur through the brachial artery (above the elbow of the arm), which carries an inherently higher risk of severe complication than catheterization via the femoral artery (due to artery size and shape). In addition, because of the lack of collateral circulation to the arm, any eventual complications during brachial catheterization may result in loss of the limb. On the other hand, vascularization in the pelvis and lower limbs is abundant, with many collateral routes to the extremities, making this access route to the abdomen inherently safer. However, there are no commercially available abdominal catheters to ensure safe and effective catheterizations through the leg (inguinal artery).

[0038] The catheter generally comprises a long hollow tube, sized to fit within the pancreatic artery, wherein the tip of the catheter has three sections, each placed at an angle to the other. The first segment, proximal to the handle, contains a length between 0.1-1.5 cm and a slope comprising between 10-40 degrees from the vertical axis, the second contains a length between 1-6 cm and a curvature such that the endpoint is on a line between 40-80 degrees from the vertical axis, and a third distal-most segment comprising a length between 0.5-3 cm, such that the endpoint of the catheter falls on a line with slope of 120-160 degrees from the vertical axis.

[0039] In preferred embodiments, if the handle to the catheter is held at 0°, the catheter tip has a first portion A closest to the handle that is about 1-2 cm long (preferably 1.45 cm) and is at about 20-30° to the handle (preferably 25-26°), a second portion B that is about 3-4 cm long and at about 65-75° to the handle (preferably at 72°), and a distal portion C that is about 0.5-1.5 cm long (preferably 1 cm), and at an angle of 130-145° (preferably at 142°). In some embodiments, the catheter tip has a constant diameter of at 1.2-1.6 mm, preferably 1.4 mm, and all segments are within 0-0.5 cm of co- planarity.

[0040] These dimensions are approximate, and the angle can be varied +/- 5° to adjust to particular patients. Further, lengths are also approximate and may need to be +/- 0.5 cm depending on patient age. Additionally, although we have illustrated the segments as straight, they may of course be curved so that the end of the segment in question falls at the desired angle.

[0041] The catheter can be made of any suitable non-toxic, biocompatible material. For example, polypropylene, polyethylene, polyether block amides, urethane elastomer, polytetrafiuoroethylene, and the like can be used. The catheter can also be made on metal, ceramic or glass or other material and coated with a biocompatible polymer, such as teflon, silicone and the like.

[0042] In one embodiment, the catheter has multiple sections with graded stiffness and the tip is softer than other parts of the catheter. The catheter can also have a hydrophilic coating, preferably a lubricious hydrophilic coating. The catheter can also have a guide wire for facilitating introduction of a flexible catheter into the body and/or a radio-opaque element for improved visualization during the procedure.

[0043] In another embodiment, the catheter has smooth transitions, made by extrusion, and in another embodiment, the catheter is re-shapeable to meet the required architecture.

[0044] Preferably, the durability and luminal changes of the catheter tip are maintained by proper selection of starting catheter materials and by minimizing surface changes and luminal irregularity so that the catheter may be re-sterilized and re-used many times. In one embodiment, the catheter has a internal root-mean-square (rms) roughness below about 29θA, and preferably below about 12θA. Li another embodiment, the cells are administered using a catheter with internal diameter of about 700 μm, and preferably below about 500 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 presents a catheter used for administration of cells and/or fluids to the pancreatic circulation.

[0046] FIG. 2 presents data on reduction of insulin usage after treatment.

45% of the patients, corresponding to 9 patients, were off insulin at one time point during the 3 years follow up. Of those 45%, one patient was off insulin during the full follow up period, 2 relapsed at the end of the follow up period, and 6 at several time points during the follow up. hi all, 9 patients were free of insulin usage for at least 6 months.

[0047] FIG. 3 presents data on AlC levels after treatment. This chart shows glycosylated Hb AlC levels. Three groups are presented with responder, partial responders and non responders and the patients in the same order as previously. For patients 12, 1, 2, and 3, the AlC values reduced rapidly and remained in the "normal" range during the following years. Some Responders actually saw an increase in AIc levels (patient 5), while others in the non-responder group showed a significant decrease in AIc, in some cases well within range for normal, suggesting that even if they did not get off insulin entirely, they were able to better manage their diabetes.

[0048] FIG. 4 presents data on peptide C levels after treatment. The left-most bars per patient are the values of C peptide before implantation. Note that all were below 0.05. Patients in the responder group had level near normal (gray shading) from 6 months post treatment up to 3 years for 6 patients out of 9. Interestingly, the level of C peptide rose also significantly in the partial responders. Patient 7 did not have any change in insulin usage, but presented a significant increase from 6 months to 3 years indicating some effect of the therapy. C peptide levels are a good marker of clinical outcome to the implantation, and this is not surprising as it is associated with regained organ function.

[0049] FIG. 5 presents data on overall results after treatment. 45% of patients were off insulin for at least one time point. 70% of patients had their C peptide levels in the "normal" range for at least 6 months indication of recovery of partial organ function, and finally, 45% of the patient had a significant reduction in their AlC level after treatment indicating better clinical management of their diabetes.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0050] We have surprisingly found that low dose mobilization of MSCs is much more effective for cell therapy than previously-used higher dosage levels. The reasons for this surprising result are not clear, but we speculate that it may be that the lower dosage allows an increase of MSC levels in the marrow, but that the dosage is not sufficiently high so as to cause mobilization into the blood stream. Thus, the number of MSCs remaining in the marrow is higher for more effective use in cell therapies.

[0051] We have exemplified herein the successful use of bone marrow cells following low dose mobilization to treat Type I diabetes, but the same low dose mobilization of bone marrow can likely be used for any cell therapy for which bone marrow and/or marrow derived stem cells have already been used, including to treat various blood disorders, Type 2 diabetes, cancers, genetic deficiencies and various autoimmune diseases. A partial list of diseases treated to date with bone marrow transplants is found in Table 1 , and new diseases are being treated all the time:

Table 1 : Diseases treated with bone marrow or stem cell transplants

Leukemias such as Acute lymphoblastic leukemia, Acute myelogenous leukemia, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, accelerated phase or blast crisis

Lymphomas such as Hodgkin's disease, Non-Hodgkin's lymphoma

Myelomas including Multiple myeloma (Kahler's disease)

Solid tumors such as Neuroblastoma, Desmoplastic small round cell tumor, Ewing's sarcoma, Choriocarcinoma

Phagocyte disorders such as Myelodysplasia

Anemias such as Paroxysmal nocturnal hemoglobinuria and Aplastic anemia (e.g., Acquired pure red cell aplasia)

Myeloproliferative disorders such as Polycythemia vera; Essential thrombocytosis; Myelofibrosis

Amyloidoses such as Amyloid light chain amyloidosis

Radiation poisoning

Lipidoses (disorders of lipid storage) such as Neuronal ceroid lipofuscinoses; Infantile neuronal ceroid lipofuscinosis (inc. Santavuori disease); Jansky-Bielschowsky disease (late infantile neuronal ceroid lipofuscinosis) and Sphingolipidoses such as Niemann-Pick disease; Gaucher disease

Leukodystrophies such as Adrenoleukodystrophy; Metachromatic leukodystrophy; Krabbe disease (globoid cell leukodystrophy)

Mucopolysaccharidoses such as Hurler syndrome; Scheie syndrome; Hurler-Scheie syndrome; Hunter syndrome; Sanfilippo syndrome; Morquio syndrome; Maroteaux-Lamy syndrome and Sly syndrome

Glycoproteinoses such as Mucolipidosis II; Fucosidosis; Aspartylglucosaminuria; Alpha- mannosidosis

Wolman disease (acid lipase deficiency)

T-cell deficiencies such as Ataxia telangiectasia, DiGeorge syndrome

Combined T- and B-cell deficiencies such as Severe combined immunodeficiency (SCID), all types

Well-defined syndromes such as Wiskott-Aldrich syndrome

Phagocyte disorders such as Kostmann syndrome; Shwachman-Diamond syndrome

Immune dysregulation diseases such as Griscelli syndrome, type II

Innate immune deficiencies such as NF-Kappa-B Essential Modulator (NEMO) deficiency

Hematologic diseases including Hemoglobinopathies; Sickle cell disease; β thalassemia major (Copley's anemia)

Anemias such as Aplastic anemia; Diamond-Blackfan anemia; Fanconi anemia

Cytopenias such as Amegakaryocytic thrombocytopenia

Hemophagocytic syndromes such as Hemophagocytic lymphohistiocytosis (HLH)

[0052] Further, although we have exemplified low dose mobilization and use of untreated marrow cells for the successful treatment of Type I diabetes where the patient is not immunosuppressed or ablated in advance, we believe that the method has general applicability, and can be used in any of the known treatment methodologies and combinations thereof, as described in Table 2.

[0053] The following examples exemplify the invention, but are not to be construed as limiting.

EXAMPLE 1. AUTOLOGOUS MARROW THERAPY FOR TYPE I DIABETES.

[0054] Patients and Methods: Twenty two patients (12 men and 10 women) were treated with total bone marrow cell implants by selective arterial catheterization. The following criteria for the inclusion of patients were used:

1. Diabetes Mellitus type 1 patients.

2. Aged between 18 and 60 years old

3. C peptide < 0.05 ng/ml.

4. Negative anti-pancreatic islet antibodies.

5. Negative anti-GAD (glutamic acid decarboxylase) antibodies. 6. Target organs not affected (renal failure with creatinine clearance below 50 ml/min, diabetic retinopathy at advanced stages, advanced arteriopathy in the lower limbs, antecedents of acute coronary syndrome, myocardial infarction or stroke, and vascular surgery).

[0055] These 22 voluntary patients were selected among 82 consulting patients. Thirty of the excluded 60 patients presented target organ lesions; 10 patients had glycated haemoglobin level over 9%; 10 patients were positive anti -pancreatic islet cell antibodies and the remaining 10 patients were under age. No diet or physical exercise program was imposed on patients.

[0056] At the time of writing this report, out of the 22 patients included in the assay, 14 were in their 16th post-implant follow-up month, 4 patients were in their 10th month, 2 patients in their six month, 2 patients in their third month. There was once a month contact with each patient, at least over the telephone, to update data and follow-up information.

[0057] An interview with the head researcher was scheduled for six and 12 post-implantation months to check on their follow-up and data in their clinical status, physical examination and daily insulin dose were recorded, including laboratory test results for blood count and kidney function. Total glycosylated haemoglobin was done prior to implantation and at 6 and 12 post-implant months. Hemaglutination of latex particles tests were taken (normal value 4.2 to 6.2 %).

[0058] Basal C peptide level was measured in peripheral blood . Levels were measured again at 6 and 12 months using an electrochemiluminiscence technique. The following results were regarded normal values in serum and plasma: 1.1-4.4 ng/ml, and 0.05 ng/mL analytical sensitivity of serum.

[0059] ICAs were measured prior to implantation and at 12 months follow- up time through quantitative indirect immunofluorescence (reference value: negative). Anti-GAD antibodies were measured by radioligand binding (reference: < 1 U/ml: negative value, and > 1.01 U/ml: positive value).

[0060] Analyzed variables were age, sex, exogenous insulin daily requirement, fasting and post-prandial glycaemia, glycated haemoglobin rate, C peptide, anti-pancreatic islet antibodies and anti-GAD follow-up levels and abdominal imaging. Variables were analysed prospectively, and non-randomized.

[0061] Materials: Bone marrow stimulation was performed with a subcutaneous 3-5 μg/kg/day Filgrastim (G-CSF) dose for 5 consecutive days. Peripheral blood flow cytometry was performed on the 6th day. We previously determined that CD 34+ cellularity levels had to be above 0.03% in peripheral blood (O.12xlO ⁸ /Kg in bone marrow) in order to schedule bone marrow extraction and catheter implantation the next day. In those cases where the CD34+ peripheral blood count was below 0.03%, the same dose of G-CSF was used for two more days, and the same was successively done until the required CD 34+ level was reached.

[0062] Cytometric analysis of the CD34+ cells was performed according to the standardized guidelines set forth in the ISHAGE (International Society of Hematotherapy and Graft Engineering) 23 protocol (Reference; Current status of CD34+ cell analysis by flow cytometry: The ISHAGE guidelines Clinical Immunology Newsletter, Volume 17, Issues 2-3, February-March 1997, Pages 21-29 by Ian Chin-Yee, Michael Keeney, Lori Anderson, Rakesh Nayar, D. Robert Sutherland). Specifically, cells were double-labeled using CD34+ and CD45 antibodies. CD45 is a pan-leukocyte marker used to increase specificity of CD34+ measures by flow cytometry. We followed the ISHAGE 23 standard protocol to quantize CD34+ cells using anti-CD45-FITC, anti-CD34-PE and PE-isotype control monoclonal antibodies.

[0063] To perform implantation, bone marrow from the posterior iliac crest was extracted. A trocar puncture was made, 60 to 80 ml of bone marrow were aspirated and mixed with sodic heparin (5.000 UI/20ml). Total bone marrow was morphologically evaluated with viability determinations (>75%), and filtered to ensure absence of blood clots, bone fragments and to ensure sterility. No cultures or in vitro enrichment procedures were performed.

[0064] Implantation Procedures: A puncture was made in the femoral artery. A guide catheter was introduced through a 5F arterial introducer, into the superior mesenteric or celiac trunk artery under radioscopic and digital angiography. The lower pancreatic artery was identified and a perfusion micro-catheter was selectively placed. The collected and filtered bone marrow cells were injected into the artery and the procedure was completed.

[0065] Selective catheterization of a target vessel is defined by successful placement of guide catheter and successful progression of micro catheter along the lower pancreatic artery, without any complications. Angiographic complications are defined as events such as embolism, thrombosis, vessel dissections or spasms, or complication of the arterial puncture process such as embolism, thrombosis, dissections, haematomas requiring surgery or blood transfusions or transfusion of blood elements. While angiographic visualization of pancreatic blush is desirable, the lack of pancreatic blush is not to be considered a complication due to angiography.

[0066] Immediate Results (22 patients): After a 3 to 5-day subcutaneous medullary stimulation with a 3-5 μg/kg/day Filgrastim (G-CSF) dose, it was observed that fifteen patients presented CD 34+ counts in peripheral blood of 0.03 to 0.06%, in four patients they were between 0.07 and 0.12%, and in three patients they were above 0.13% and up to 0.22%.

[0067] Angiographic success was achieved in all implanted patients, dominance of hepatic artery was observed in 20 out of 22 patients and in the other two patients there was dominance in the superior mesenteric artery. Pancreatic blush was observed after injection of bone marrow collection in 12 patients. No vascular complications in the dominant artery were observed. Average time of procedure was 16 minutes (10-30 min. range). No patient evidenced adverse events during the intra-hospital period. Average length of hospitalization time was 12 hrs. (6 - 24 h. range).

[0068] 30-day Results (22 patients): Twelve patients reported hypoglycaemia episodes (symptoms were compatible with glycaemias below 60 mg/dl) that were repeated (occuring for at least three consecutive days), and not attributable to common causes such as high insulin dose, diet, strenuous exercise, etc. This resulted in a decrease of total daily insulin usage (20% on average). No changes were observed in terms of general physical condition, physical examination, and laboratory controls of blood count and renal function were normal in all patients. No adverse events were observed.

[0069] 6-month Results (18 patients): None of the treated patients changed body weight by over 10% of weight prior to implantation. Four patients attained total suppression of daily insulin dose, normal basal and stimulated C peptide values, fasting and post prandial glycaemia and glycated haemoglobin levels. No blood count and renal function laboratory test alterations were observed.

[0070] Follow-up of the remaining 12 patients showed decrease in total daily insulin dose and increase in basal C peptide values (although not reaching normal values), without significant changes in glycated haemoglobin levels.

[0071] Two patients belonging to this group did not present significant clinical or laboratory test changes. No adverse events were observed.

[0072] 12-month Results (14 patients): None of the treated patients changed body weight by over 10% of weight prior to implantation. Six patients attained total suppression of daily insulin dose, normal basal C peptide values, fasting and post prandial glycaemia and glycated haemoglobin levels. No blood count and renal function laboratory test changes were observed.

[0073] Three out of these 6 patients had been insulin-free for over 6 months

(average 8 months, within a 6-12 range) and 3 patients for less than 6 months (average 3 months, within a 2-5 range).

[0074] After 4 months of total insulin suppression, one patient had to resume pre-implantation administration level. No blood count and renal function alterations (urea and creatinine in blood tests) were observed.

[0075] Six patients were administered less than 66% of the initial daily insulin dose and showed increase in basal C peptide values (although not reaching normal values), with no significant changes in glycated haemoglobin levels, and one patient was administered less than 50% of insulin dose. One patient, who at 120 days was a member of the implanted group, gradually resumed the insulin dose required prior to implantation.

[0076] Two patients never showed significant post-implant changes. No adverse events were observed.

[0077] Glycaemia Level Follow-up: Patients took daily fasting and postprandial glycaemia self-tests at least twice during the post-implantation period. It was observed that 18 out of the 22 patients had symptomatic hypoglycaemic episodes not related to other causes (42 mg/dl average, 20 to 58 mg/dl range), at different times during the day and at least for three consecutive days before decreasing their insulin administration. Such hypoglycaemic episodes were registered as of the first month of follow-up and up to a year of follow-up and were more frequent between the fourth and eighth month after stem cell transplantation. It was observed that hypoglycaemic episodes were always symptomatic, not serious. It was also observed that as of the first month of follow-up, hyperglycaemic episodes (glycaemia above 200 mg/dl any time during the day) were infrequent and it led patients to stop using fast-acting insulin.

[0078] Glycated Haemoglobin Follow-up: At one follow-up year, patients who had managed to suppress exogenous insulin administration and reach normal basal and stimulated C peptide levels, showed a decrease in glycated haemoglobin value until it became normal. Patients who had not managed to suppress exogenous insulin administration or reach normal C peptide levels, showed glycated haemoglobin levels similar to those obtained prior to implantation with a 1% variation in some cases.

[0079] C Peptide Level Follow-up: It was observed that patients who had attained total suppression of insulin administration reached normal levels of basal and stimulated C peptide. Patients who had decreased their daily insulin administration dose by over 66% showed a C-peptide level increase at least three times higher than that obtained prior to implantation, although they had not reached normal values. No significant changes in C peptide level were observed in patients who had decreased their daily insulin administration dose by 50%, or in patients that showed no clinical changes.

[0080] Anti-Pancreatic Islet Antibodies Follow-up: It was observed that all patients showed negative anti-pancreatic islet antibodies at one year of bone marrow stem cell implantation follow-up.

[0081] The efficacy of the therapy regime and the function of the implanted bone marrow cells may be attributable to various specific functions of the cells. Specifically, mobilizing marrow stem cells to peripheral blood may contribute to systemic immune suppression. Additionally, local delivery of bone marrow has been shown to encourage local neo-vascularization, which may serve to further aliment the pancreatic tissue. Based on increased C-peptide levels observed in 70% of patients, some organ function is reached, which may be attributable to transdifferentiation events and/or local action of cytokines and chemokines signaling to local resident progentiors and other cells able to respond to paracrine factors released by the cells.

[0082] 24-months results (25 patients) Five patients achieved a complete cessation of the daily insulin, achieved normalization the basal C-peptide values, fasting glucose and post prandial and glycated hemoglobin (No. 1, 2, 4, 9 and 12). Three patients, after an average of 9 months of suppression of insulin dose returned to the pre-implant (No. 3, 5 and 18). No significant alterations in blood count or renal function laboratory (measurement of blood urea and creatinine). Three patients received less than 66% of the initial daily insulin dose with elevated basal c-peptide values (although not to normal values), without significant changes in the measurement of glycated hemoglobin, (No. 6, 11 and 15). Of this group two patients returned to using pre-implant dose of insulin gradually.

Nine patients are receiving the same amount of insulin pre-implant with abnormally decreased peptide c (No. 3, 5, 7, 8, 10, 13, 14, 16, 17 and 24). A single patient (No. 7) never produced significant changes post-implant. There were no adverse events.

[0083] 36-months results (20 patients) Three patients had maintained complete elimination of daily insulin dose, normalized the basal C-peptide values, fasting glucose and post prandial and glycated AlC hemoglobin levels (No. 1, 12 and 19).

[0084] Three patients, after an average of 9 months of suppression of insulin dose returned to the pre-implant (No. 3, 5 and 18). No significant alterations in blood count or renal function laboratory (measurement of blood urea and creatinine).

[0085] Three patients were using less than 66% of the initial daily insulin dose with elevated basal c-peptide values (although not to normal values), without significant changes in the measurement of glycated hemoglobin, (No. 6, 11 and 15). Of this group two patients returned to using pre-implant dose of insulin gradually.

[0086] Nine patients are receiving the same amount of insulin pre-implant with abnormally decreased peptide c (No. 3, 5, 7, 8, 10, 13, 14, 16 and 17).

[0087] A single patient (No. 7) never produced significant changes post- implant. No adverse events were observed. [0088] Evolution of blood glucose. The patients underwent daily blood glucose self-analysis and post-prandial fasting at least twice. It was noted that patients had episodes of symptomatic hypoglycemia, unrelated to other causes (42 mg / dl average range of 20 to 58 mg / dl), at different times of day and at least three consecutive days before of lower doses of insulin. Episodes of hypoglycemia were found from the first month, and until follow-up, occurring more frequently between the fourth and eighth month of evolution. It was noted that the hypoglycemic episodes were always symptomatic, and never serious. It was also observed from the month of evolution that episodes of hyperglycemia (blood glucose above 200 mg / dL at any time of day) were rare prompting patients to abandon the use of rapid-acting insulin.

[0089] Evolution of C-peptide. In the responder patient group (patients who ceased exogenous insulin usage), peptide C levels were normalized. In patients who decreased the daily insulin dose by more than 66% was an increase of at least three times the value obtained prior to implant, although they failed to fully normalize. In patients decreased by 50% or less of the starting insulin use amounts (non-responders), these patients showed no significant changes in the C-peptide.

[0090] Evolution of glycated hemoglobin AlC Levels. In patients who were able to eliminate exogenous insulin use and normalized basal and stimulated C- peptide, AlC levels decreased substantially. In patients the non-responder and partial responder group, and failed to normalize C-peptide levels, glycosylated hemoglobin values were similar to those obtained prior to the implant with a variation of 1% in some cases.

[0091] Evolution of islet auto-antibodies. All patients remain negative for antibodies a year, two and three years of evolution of their implants bone marrow stem cells.

[0092] Patients that are insulin-free with normal C-peptide and AlC levels

[0093] Overall, 45% of patients were off insulin for at least one time point.

70% of patients had their C peptide levels in the "normal" range for at least 6 months indication of recovery of partial organ function, and finally, 45% of the patient had a significant reduction in their AlC level after treatment indicating better clinical management of their diabetes.

EXAMPLE 2. CATHETERIZATION AND IRRIGATION.

[0094] Patients suffering from Type 1 diabetes are subjected to catheterization via a puncture made in the femoral artery. Guide catheter measuring 5F and side holed sheath introducers, are placed in the superior mesenteric or celiac trunk artery under radioscopic and digital angiography. The lower pancreatic artery is identified and a perfusion micro-catheter is selectively placed and cell-free buffer solution injected in the artery. Thus the implant procedure is completed. Selective catheterization of the affected vessel (selective placement of guide catheter and successful progression of micro catheter along lower pancreatic artery) is regarded an angiographic success without vascular or arterial puncture complications (embolism, thrombosis, dissections, spasms, or haematomas requiring surgery or blood transfusions or transfusion of blood elements), with or without pancreatic blush (pancreatic blush refers to good microvascular visualization during angiography of the pancreatic parenchyma). EXAMPLE 3. ACTIVATED MARROW FOR CARDIAC THERAPY

[0095] Bone marrow stimulation with a subcutaneous 3-5 μg/kg/day

Filgrastim (granulocyte colony stimulating factor G-CSF) dose for 5 consecutive days is performed on patients participating in the trial. A peripheral blood flow cytometry is taken on the 6th day. It is predetermined that CD 34+ cellularity had to be above 0.03% in peripheral blood (0.12xl0 ⁸ /Kg in bone marrow) to schedule bone marrow extraction and catheter implantation the next day. In cases when peripheral blood count is below the predetermined one, the same dose of G-CSF is used for two more days, and the same is successively done until the previously determined CD 34+ number is reached.

[0096] Blood flow cytometry determined a homogeneous population of

CD34+ cells compared with the number of CD45+ cells. ISHAGE 23 is the protocol used to quantize CD34+ cells using anti-CD45-FITC, anti-CD34-PE and isotyρe-PE control monoclonal antibodies.

[0097] Bone marrow from the posterior iliac crest is extracted. A trocar puncture is made, 60 to 80 ml of bone marrow is aspirated and mixed with sodic heparin (5.000 UI/20ml). Filtered bone marrow is morphologically evaluated for feasibility determinations, absence of blood clots, bone residue and bacteria. No cultures or in vitro enrichment procedures are performed. The bone marrow is administered within the heart (intramyocardial) and coronary artery (intracoronary) tissues of heart disease patients utilizing the investigational MYOSTAR Injection Catheter, researchers administer bone marrow-derived stem cells into patients' left heart ventricle, relying on the NOGA System to aid in accurately identifying the target injection site.

EXAMPLE 4. MARROW AND HUCPVC CELLS FOR DIABETES

[0098] Bone marrow stimulation with a subcutaneous 3-5 μg/kg/day

Filgrastim (granulocyte colony stimulating factor G-CSF) dose for 5 consecutive days is performed on patients participating in the trial. A peripheral blood flow cytometry count is performed on the 6th day. It is predetermined that CD 34+ cellularity has to be above 0.03% in peripheral blood (0.12x10 ⁸ ZKg in bone marrow) to schedule bone marrow extraction and catheter implantation the next day. hi cases when peripheral blood count is below the predetermined one, the same dose is used for two more days, and the same is successively done until the previously determined CD 34+ number is reached.

[0099] Blood flow cytometry determines a homogeneous population of

CD34+ cells compared with the number of CD45+ cells. ISHAGE 23 is the protocol used to quantize CD34+ cells using anti-CD45-FITC, anti-CD34-PE and isotype-PE control monoclonal antibodies.

[00100] Bone marrow from the posterior iliac crest is extracted. A trocar puncture is made, 60 to 80 ml of bone marrow are aspirated and mixed with sodic heparin (5.000 UI/20ml). Filtered bone marrow is morphologically evaluated for feasibility determinations, absence of blood clots, bone residue and bacteria. No cultures or in vitro enrichment procedures are performed. The bone marrow cells are mixed with an equal number of HUCPVC cells and administered intravenously to patients with Type I diabetes.

EXAMPLE 5. MARROW AND TARGETING AGENT FOR DIABETES

[00101] Bone marrow stimulation with a subcutaneous 3-5 μg/kg/day

Filgrastim (granulocyte colony stimulating factor G-CSF) dose for 5 consecutive days is performed on Charcot-Marie-Tooth disease patients participating in a trial. A peripheral blood flow cytometry is taken on the 6th day. It is predetermined that CD 34+ cellularity has to be above 0.03% in peripheral blood (0.12xl0 ⁸ /Kg in bone marrow) to schedule bone marrow extraction and catheter implantation the next day. In cases when peripheral blood count is below the predetermined one, the same dose is used for two more days, and the same is successively done until the previously determined CD 34+ number is reached.

Blood flow cytometry determines a homogeneous population of CD34+ cells compared with the number of CD45+ cells. ISHAGE 23 is the protocol used to quantize CD34+ cells using anti-CD45-FITC, anti-CD34-PE and isotype-PE control monoclonal antibodies. Bone marrow from the posterior iliac crest is extracted. A trocar puncture is made, 60 to 80 ml of bone marrow are aspirated and mixed with sodic heparin (5.000 UI/20ml). Filtered bone marrow is morphologically evaluated for feasibility determinations, absence of blood clots, bone residue and bacteria. No cultures or in vitro enrichment procedures are performed. The bone marrow is administered along with an affinity targeting agent (a bispecific antibody) into the pancreatic circulation of patients with type I diabetes.

EXAMPLE 6: CATHETER

[00102] Diagnostic catheterization of the pancreas is rarely performed mainly due to the fact that the pathologies that would require it are rare, and that other more sensitive, specific and noninvasive diagnostic methods (e.g., CT, MRI, ultrasound) exist. However, when required, 90% of these catheterizations are performed via the brachial artery, which is more complex and can result in greater complications than access via the femoral artery. This finding is supported in the medical literature. A catheter designed to allow cell implantation, whether by brachial or femoral route, does not exist because the procedure itself does not yet exist. Thus, there are no catheters on the market designed for cell implants in pancreatic and hepatic circulation.

[00103] Existing catheters are designed for other things like performing diagnostic studies. These angiographic studies are simple and require no specific features in the catheter. To avoid complexities and complications in delivering cells, we sought to perform the procedure via the femoral artery, hi the first patient case we attempted catheterization via femoral artery with a Multi Purpose catheter. However, since the distal segment of the catheter is very long, selective catheterization of the mesenteric trunk was very difficult. Even when successful, the very long tip segment entered the splenic or hepatic artery, eliminating the ability to control the catheter.

[00104] In an attempt to correct these problems we tried to make an intermediate curve in the distal segment of a multi-purpose catheter. When we tried to put it in the celiac trunk, we noticed that this tip angle greatly shortened the distal segment length. This was problematic because we could never fully enter the mesenteric trunk with the catheter, the catheter was never steady in the vessel, and there was a great amount of reflux of contrast agent into the aorta.

[00105] For this reason we tried with another type of catheter, one intended for Mammary Aorto Coronary bypass via the left breast. With this catheter as originally designed, it is impossible to perform selective catheterization of the celiac trunk. However, its design did allow the introduction of the three curves and segments of our own design. For regulatory and ethical reasons, we modified this currently available straight catheter in the following way in order to minimize risks to the patient and ensure successful release of the cell sample: We created a mold with the required curvatures and dimensions (see below), determined empirically and by knowledge of the related anatomy. Said mold was then heated, the catheter was placed within the mold and after a few minutes, the catheter tip assumed the shape of the mold. The catheter was then sent for sterilization (ethylene oxide).

[00106] The above-mentioned catheter has a very curved distal segment, due to the anatomical position of where the left mammary artery originates, therefore we tried to adjust to widen the angle of curvature. This was successfully achieved but the catheter was short and was not selective. For this reason, we further modified the first angle in the body of the catheter that was formerly straight. With this modification, the catheter was then sufficiently long to successfully perform selective catheterization. Furthermore, since the distal segment was short, it was possible to safely maneuver the catheter within the mesenteric trunk to guide the catheter into the hepatic circulation, and place within it the cerebral microcatheter.

[00107] The catheter generally comprises a long hollow tube sized to fit within an artery. In the final design, shown in Figure 1, the tip of the catheter that has three sections, each placed at an angle to the other. In preferred embodiments, if the handle to the catheter is held at 0°, the catheter tip has a first portion A closest to the handle that is about 1-2 cm long (preferably 1.45 cm) and is at about 20-30° to the handle (preferably 25- 26°), a second portion B that is about 3-4 cm long and at about 65-75° to the handle (preferably at 70-72°), and a distal portion C that is about 0.5-1.5 cm long (preferably 1 cm), and at an angle of 120-160° (preferably at about 140-142°). The segment may be straight or more or less curved (not shown), so long as the distal most end of the segment lies at the degree from vertical required.

[00108] The catheter was used in 20 patients, was simple, safe, effective and rapid. We did not observe complications due to catheterization and the outcome was 100% successful (implant in the appropriate vessel).

[00109] What is claimed is: