Patent Application
20130095081
The discovery of pluripotent cells in the adipose tissue has revealed a novel source of cells that may be used for autologous cell therapy to regenerate tissue. The pluripotent cells reside in the “stromal” or “non-adipocyte” fraction of the adipose tissue; they were previously considered to be pre-adipocytes, i.e. adipocyte progenitor cells, however recent data suggest a much wider differentiation potential. Zuk et al. were able to establish differentiation of such subcutaneous human adipose stromal cells, or adipose stem cell cells as referred herein, (“ASCs”) in vitro into adipocytes, chondrocytes and myocytes. These findings were extended in a study by Erickson et al., which showed that human ASCs could differentiate in vivo into chondrocytes following transplantation into immune-deficient mice. More recently, it was demonstrated that human ASCs were able to differentiate into neuronal cells, osteoblasts cardiomyocyte, and endothelial cells.
EPCs (with a range of phenotypic definitions) have been studied extensively over the past decade since their original isolation from adult peripheral blood and, later from bone marrow, umbilical cord blood, vessel wall. Umbilical cord blood contains a population of EPC with a particularly high proliferative potential, termed endothelial colony forming cells (“ECFCs”). Recently, it was shown that ECFCs immobilized in matrices form functional vessels in vivo when implanted in mice. While the presence of blood cells within the capillary networks formed by such human EPCs confirmed anastomoses with host vasculature, the neo-vessels were limited in frequency and size. This finding is similar to a prior study with implants containing fully mature endothelial cells (“ECs”), in which vessels were narrow-caliber and comprised of a single layer of cells. In the latter study, large caliber vessels with thick walls were formed only with ECs overexpressing the bcl-2 oncogene, presumably as a consequence of repressed EC apoptosis as well as augmented recruitment of host mesenchymal cells. With non-transformed ECs, the failure to establish stable, mature vasculature may be due to prolonged absence of a stabilizing layer of mural cells, which include pericytes and smooth muscle cells (“SMCs”). Although EPCs secrete multiple angiogenic factors to attract perivascular cells, conditions created in the matrix implants in vivo may restrict recruitment and, thereby, fail to prevent disassembly of vessels due to EC apoptosis. It has been demonstrated that human saphenous vein and aortic smooth muscle cells, blood derived and bone marrow MSC cooperate with ECs to promote stable vascular networks. However, the utility of these findings is restricted by the scarcity of adequate and easily-accessible sources of these perivascular/mural cell types.
Diabetes mellitus is a highly prevalent disease, afflicting more than 10% of the US population greater than 20 years of age; and more than 23% of the population greater than 60 years old (NIH-NIDDK statistics from 2007). Type 1 diabetes mellitus (T1DM) accounts for about 10% of diabetes, and results from a cascade of events that culminates in destruction of the insulin-producing β cells of the islets of Langerhans. These events are initiated after a nonspecific injury to the β0 cell results in exposure of autoantigens, after which macrophages and other antigen presenting cells activate CD4+ and CD8+ T cells. A complex and destructive interplay ensues, which is amplified by the secretion of proinflammatory cytokines such as interleukin 1β (IL-1β), tumor necrosis factor a (TNFα), and interferon γ (IFN γ) from macrophages, T cells, and the β cell itself, enabling “a vicious cycle” of necrotic and apoptotic β cell death.
Type 2 diabetes mellitus (T2DM) results from a combination of peripheral insulin resistance and β-cell dysfunction. In the initial phases of disease, the β cell is able to compensate for the insulin resistance by increasing insulin production, resulting in hyperinsulinemia. However, this compensation is limited in time; as the β cell function begins to fail as a result of increasing metabolic demands, the insulin levels fall. The United Kingdom Prospective Diabetes Study demonstrated that patients with T2DM experience progressive β cell dysfunction despite most drug treatments to lower blood glucose; this dysfunction is characterized by profound insulin secretory defects. Clinically, this manifests as a loss of the first-phase response to intravenous glucose, delayed and blunted insulin responses to ingestion of a mixed meal, and loss of the normal pattern of insulin secretion. In T2DM, the fluid milieu of the body typically exhibits hyperglycemia (again, expressed over time in terms of HbA1c) in the context of hyperinsulinemia.
While there are important differences in the underlying pathophysiology of the two forms of diabetes, β cell failure remains at the core of both Type 1 and Type 2 diabetes. Likewise, treatments that successfully employ β cell replacement could have utility in T1DM and T2DM.
In 2000, Shapiro et al. published a highly promising account of islet transplantation at the University of Alberta (Edmonton, Canada), where 7 out of 7 patients with T1DM who were treated with islet transplantation remained insulin-independent after one year. Tremendous interest in advancing the field of islet transplantation ensued. However to date, long term results from islet transplants have been somewhat disappointing, largely due to islet graft failure. Several reasons for the graft failure have been proposed, including inadequate graft mass due to acute inflammatory destruction; problems with islet quality, viability, and engraftment; auto and allo-immune destruction; and inadequate or abnormal revascularization of the islet graft. The normal islet is highly vascular with approximately 10 times more blood delivered to the endocrine pancreas as compared to exocrine tissue. This is an impressive discrepancy given the consideration that endocrine cells comprise only 1-2% of the total mass of the pancreas. Normally, a central arteriole supplies blood to the islet through a highly fenestrated capillary network, and this native vascular system is disrupted during the isolation procedure.
A treatment capable of increasing the efficacy of islet cell transplantation, or otherwise promoting glucose homeostasis in a mammal would be greatly appreciated.
Disclosed herein are various methods and compositions for treating a patient having a disorder. At least some of the methods and compositions involve the use of mammalian stem cells, such as mammalian adipose stem cells, to treat the disorder.
In at least one embodiment of a method for treating a patient with a disorder, such as an insulin-related disorder, the method comprises the step of administering a cell-based composition to a patient with an insulin-related disorder to treat the insulin-related disorder, the cell-based composition comprising at least one mammalian stem cell and optionally at least one islet cell, the at least one mammalian stem cell capable of prolonging an effective life of the at least one islet cell. The step of administering a cell-based composition, in at least one embodiment of the method, comprises administering the cell-based composition comprising at least one mammalian adipose stem cell, and optionally at least one mammalian endothelial or endothelial progenitor stem cell. Further, the at least one mammalian stem cell of the cell-based composition administered to the patient may previously have been isolated from the patient. Moreover, administering the cell-based composition to a patient comprise a method of administration selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, retrograde venous injection, arterial injection, and surgical implantation.
In at least one embodiment of a method for treating a patient with an insulin-related disorder, the insulin-related disorder is selected from the group consisting of Type 1 diabetes, Type 2 diabetes, gestational diabetes, pre-diabetes, and impaired glucose tolerance. Additionally, the cell-based composition may further comprise at least one islet cell and/or a biological agent capable of promoting cell growth. The biological agent may comprise a growth factor, including but not limited to a hepatocyte growth factor, an insulin-like growth factor, a fibroblast growth factor, and a vascular endothelial growth factor. The biological agent may also be an anti-apoptotic agent or a pro-angiogenic agent. Additionally, in at least one embodiment of the method, the cell-based composition is provided in a form selected from the group consisting of a matrix form and a capsule form.
The administration step, in at least one embodiment of the method of treating a patient, is performed to treat an insulin-based disorder by promoting production of insulin within the patient. Further, the administration step may be performed to treat the insulin-based disorder by at least one of (1) reducing a rate of peripheral insulin resistance within the patient, (2) reducing a rate of β-cell dysfunction within the patient, and (3) increasing the patient's glucose tolerance.
In at least one embodiment of the method or composition of the present disclosure, the at least one mammalian stem cell is selected from the group consisting of at least one a CD10+ mammalian adipose stem cell, at least one a CD13+ mammalian adipose stem cell, at least one a CD34+ mammalian adipose stem cell, at least one a CD34− mammalian adipose stem cell, at least one a CD45+ mammalian adipose stem cell, at least one a CD45− mammalian adipose stem cell, at least one a CD90+ mammalian adipose stem cell, at least one a CD90− mammalian adipose stem cell, at least one a CD 140a+ mammalian adipose stem cell, at least one a CD140a− mammalian adipose stem cell, at least one a CD140b+ mammalian adipose stem cell, and at least one a CD140b− mammalian adipose stem cell.
In at least one embodiment of a method for treating a patient with a disorder, the cell-based composition further comprises at least one endothelial cell. Additionally, in at least one embodiment, the ratio of the at least one mammalian stem cell to the at least one endothelial cell is selected from the group consisting of at least about 8 to about 1, about 4 to about 1, about 2 to about 1, about 1 to about 1, about 1 to about 2, about 1 to about 4, about 1 to at least about 8.
In at least one embodiment of a method for treating a patient with an insulin-related disorder, the method comprises the step of administering a cell-based composition to a patient with an insulin-related disorder to treat the insulin-related disorder, the cell-based composition comprising at least one mammalian stem cell and at least one islet cell, the at least one mammalian stem cell capable of prolonging an effective life of the at least one islet cell and further capable of effectuating promotion of insulin production within the patient. Further, the cell-based composition may also comprises a biological agent capable of promoting cell growth, the biological agent selected from the group consisting of a hepatocyte growth factor, an insulin-like growth factor, a fibroblast growth factor, a vascular endothelial growth factor, an anti-apoptotic agent and a pro-angiogenic agent.
In at least one embodiment of a method for treating a patient with a disorder, the method comprises the step of administering a cell-based composition to a patient with a disorder to treat the disorder, the cell-based composition comprising at least one mammalian stem cell and optionally at least one islet cell, the at least one mammalian stem cell capable of prolonging an effective life of the at least one islet cell. The step of administering a cell-based composition in at least one embodiment of the method of treatment comprises administering the cell-based composition to a patient with an insulin-related disorder, where the insulin-related disorder may be selected from the group consisting of Type 1 diabetes, Type 2 diabetes, gestational diabetes, pre-diabetes, and impaired glucose tolerance.
In at least one embodiment of a method for treating a patient with an insulin-related disorder, the method comprises the step of administering a cell-based composition to a patient with an insulin-related disorder to treat the insulin-related disorder, the cell-based composition comprising at least one mammalian stem cell capable of effectuating promotion of insulin production within the patient.
In at least one embodiment of a cell-based composition of the present disclosure, the cell-based composition comprises at least one mammalian stem cell, and optionally at least one islet cell, wherein the at least one mammalian stem cell capable of prolonging an effective life of the at least one islet cell, and wherein the composition is effective to treat a patient with an insulin-related disorder by effectuating the promotion of insulin production within the patient. Optionally, the cell based composition may further comprise at least one islet cell and/or a biological agent capable of promoting cell growth. The biological agent may comprise a growth factor, including but not limited to a hepatocyte growth factor, an insulin-like growth factor, a fibroblast growth factor, and a vascular endothelial growth factor. Further, the biological agent may be selected from the group consisting of an anti-apoptotic agent and a pro-angiogenic agent.
In at least one embodiment of the cell-based composition, the cell-based composition is provided in a form selected from the group consisting of a matrix form and a capsule form. Additionally, the cell-based composition may further comprise a biologically-compatible carrier. Further, the cell-based composition may be effective to treat the patient by at least one of (1) reducing a rate of peripheral insulin resistance within the patient, (2) reducing a rate of β-cell dysfunction within the patient, (3) increasing the patient's glucose tolerance. In at least one embodiment of the composition, the at least one mammalian stem cell comprises at least one mammalian adipose stem cell.
In at least one embodiment of a cell-based composition of the present disclosure, the cell-based composition comprises at least one islet cell, and at least one mammalian stem cell capable of prolonging an effective life of the at least one islet cell, wherein the composition is effective to treat a patient with an insulin-related disorder by effectuating the promotion of insulin production within the patient. Additionally, an embodiment of the cell-based composition may further comprise a biological agent capable of promoting cell growth, the biological agent selected from the group consisting of a hepatocyte growth factor, an insulin-like growth factor, a fibroblast growth factor, a vascular endothelial growth factor, an anti-apoptotic agent and a pro-angiogenic agent.
In at least one embodiment of a method of producing a cell-based composition useful to treat a patient, the method comprises the steps of isolating at least one mammalian stem cell from a mammal, optionally expanding the at least one mammalian stem cell to produce a plurality of mammalian stem cells, and combining at least some of the plurality of mammalian stem cells or the isolated at least one mammalian stem cell with at least one islet cell to form a cell-based composition effective to treat a disorder of a patient. The at least one mammalian stem cell may in at least one embodiment comprise at least one mammalian adipose stem cell. Additionally, an embodiment of the method of producing a cell-based composition may further comprise the step of administering the cell-based composition to the patient to treat the disorder, including but not limited to Type 1 diabetes, Type 2 diabetes, gestational diabetes, pre-diabetes, and impaired glucose tolerance. Further, the mammal in an embodiment of the method of producing a cell-based composition may be the patient. Additionally, the step of expanding may comprise expanding the at least one mammalian stem cell in a cell culture environment to produce the plurality of mammalian stem cells.
In at least one embodiment of a method of vascularizing tissue, the method comprises the steps of combining at least one mammalian stem cell with a plurality of endothelial cells and a matrix to create a vascularization composition, and administering the vascularization composition to a patient, wherein the vascularization composition is useful to increase vessel formation at the site of administration. The at least one mammalian cell of an embodiment of a method or composition of the present disclosure may be VEGF+ and HGF+, and may further be from the patient. Further, the at least one mammalian stem cell comprises at least one adipose stem cell.
In at least one embodiment of a method of treating a patient, the step of administering effectuates vascularization of a tissue of the patient at or near the site of administration of the cell-based composition.
In at least one embodiment of a method to determine the effectiveness of a cell-based composition to treat a mammalian disorder, the method comprises the steps of placing at least one mammalian stem cell in a first cell vessel, placing at least one islet cell in the first cell vessel with the at least one mammalian stem cell, placing an additional at least one mammalian stem cell in a second vessel that does not contain a mammalian stem cell, and comparing a selective morbidity of the at least one islet cell in the first vessel and the second vessel, and wherein the comparison is indicative of an ability of the at least one mammalian stem cell to prolong an effective life of the at least one islet cell in the first cell vessel, which is indicative of an effectiveness of the at least one mammalian stem cell to treat a mammalian disorder, Optionally, the step of comparing the selective morbidity may also comprise the step of determining the selective morbidity of the at least one islet cell in the first vessel and the second vessel with a diagnostic agent, such as an antibody, a reactive chemical compound, and a labeled molecule.
a-h show the histological analysis of human adipose tissue, according to at least one embodiment of the present disclosure;
a-e show visual (panels a-c), graphical (panel d), and histological (panel e) depictions of the synergy between ASCs and ECs in promoting vasculogenesis, according to at least one embodiment of the present disclosure;
a-d show visual (panels a-c) and graphical (panel d) representations of the effect of ASC deficient in HGF on relative perfusion rates in ischemic limbs, according to at least one embodiment of the present disclosure;
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.
The disclosure of the present application provides various methods for cell-based therapies. For instance, at least some embodiments of the methods disclosed herein heighten the treatment potential of islet cells. Due to the failure of islet cell grafts for the treatment of Type I and Type II diabetes, and considering the increasing level of diabetes among the general population, there is need for a method of treating diabetes.
Adipose stem cells (ASCs) are isolated from human, and other mammalian, subcutaneous adipose tissue according to the method of Zuk et al. ASCs are predominantly localized in the peri-endothelial layer of the vessels in vivo (in adipose tissue), and are phenotypically and functionally equivalent to pericytes associated with microvessels. The ASCs may, in at least one illustrative example, be isolated at a level of about 108 cells per 100 ml of lipoaspirate. Further, following isolation, the isolated ASCs may be cultured on tissue culture plastic in EGM-2mv medium. In this medium, ASC can expand to about 1000-fold over a 10 day period. Further, ASCs isolated from humans (hASCs) routinely secrete a wide variety of bioactive molecules, such as VEGF, HGF, and GM-CSF, which participate in stimulation of EC survival and proliferation and stabilization of endothelial networks formed on the surface of Matrigel.
Referring to
According to an exemplary cell-based composition of the present disclosure, the composition further comprises a biological agent. The biological agent, in at least one exemplary embodiment, is selected from a group consisting of hepatocyte growth factor, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor, an anti-apoptotic agent, and a pro-angiogenic agent. Treatment of the at least one mammalian stem cell with a biological agent may be for a period of at least about one minute, at least about twelve hours, at least about twenty-four hours, at least about forty-eight hours, or at least about 72 hours. Optionally, an embodiment of a cell-based composition may also comprise at least one endothelial cell. Moreover, an exemplary cell-based composition may further be provided in a form, such as a matrix form and a capsule form. The form, in at least one example, may comprise collagen, fibronectin, a combination thereof, or any acceptable and biocompatible form,
In at least one embodiment of the method 100 of treating a patient, the patient has an insulin-based disorder, such as Type 1 diabetes, Type II diabetes, or gestational diabetes. Further, the step of administering the cell-based composition to the patient treats the patient's insulin-based disorder.
According to at least one embodiment of an cell-based composition of the present disclosure, the composition comprises at least one islet cell, and at least one adipose stem cell, wherein the at least one adipose stem cell is capable of repressing cell death of the at least one islet cell. Optionally, the composition may further comprise at least one endothelial cell. Further, an cell-based composition may also comprise a biological agent selected from a group consisting of hepatocyte growth factor, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor, an anti-apoptotic agent, and a pro-angiogenic agent.
According to at least one embodiment of the present disclosure, combining the at least one islet cell with at least one mammalian stem cell, that may be an adipose stem cell, and optionally at least one endothelial cell, generates a cell-based composition that is capable of at least one of (1) promoting the production of insulin within the patient when introduced, (2) reducing the rate of peripheral insulin resistance within the patient when introduced, (3) reducing the rate of β-cell dysfunction within the patient when introduced, and (4) increasing the patient's glucose tolerance when introduced. Additionally, in at least one embodiment, the administering of the cell-based composition to a patient increases vascular blood flow at the site of administration, and increases the level of blood glucose control of the patient.
Referring to
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Labeled molecules, for use as indicator compounds, may be any molecule that absorbs, excites, or modifies radiation, such as the absorption of light (e.g. dyes and chromophores) and the emission of light after excitation (fluorescence from flurochromes). Additionally, labeled molecules may have an enzymatic activity, by which it catalyzes a reaction between chemicals in the near environment of the labeling molecules, producing a signal which include production of light (chemi-luminescence) or precipitation of chromophors, dyes, or a precipitate that can be detected by an additional layer of detection molecules.
Exemplary fluorescence labels may produce the presence of light at a single wavelength, or a shift in wavelengths.
A majority of human ASCs (hASCs) isolated as described Zuk et al. and additionally enriched by attachment to tissue culture plastic, express the stem cell marker CD34 (in the first days of culture), as well as co-express several mesenchymal cell markers (CD10+/CD13+/CD90+) and pericyte markers (CD140a+/CD140b+/NG2+) (
Following the identification of ASC markers, the location of ASC in adipose tissue was determined in situ by immunochemical staining. Staining for CD34 (
To address ASC-EC interactions, endothelial progenitor cells (EPCs) were isolated and expanded from umbilical cord vein blood of healthy newborns. Isolated mononuclear cells (MNC) were cultured on collagen-coated plastic in EGM-2/10% FBS. Cells were expanded and utilized up to passage 6, without significant changes in cell morphology, markers, and responses to factor stimuli). Throughout the work presented herein, EPCs derived from cord blood by this technique were used, to maximize consistency.
To evaluate the effect of factors secreted by hASCs on ECs, human microvascular ECs (HmVEC) cultured in growth factor-free media were exposed to conditioned media (CM) of hASCs incubated for 72 hours in either normoxic or hypoxic conditions (
To understand a broader range of factors that could additionally participate in the effect on EC, ASC-normoxic CM (72 hours) was evaluated using RayBio Cytokine Antibody Arrays (RayBiotech Inc).
Culturing endothelial cells on tissue culture plastic alone or coated with extracellular matrix proteins results in cell expansion to a monolayer. To examine the functional interaction of ASC and EC, EC were plated on a monolayer of ASC directly on plastic, without the addition of exogenous ECM proteins. Co-culture under these conditions promotes spontaneous assembly of EC into vascular networks over 3-6 days (
To extend the in vitro finding that ASC promote endothelial cord formation and stability, an in vivo model was employed by embedding ASC together with EPC in 3D collagen gels, followed by their implantation subcutaneously into NOD/SCID mice for fourteen days. Gross evaluation of implants, following the fourteen day period, showed an obvious difference between the appearance of gels containing ASC or EPC alone (white, minimally attached to adjacent host tissue) and gels containing their mixture, which were routinely blood-filled and tightly connected to the mouse abdominal wall (
To permit serial assessment of vascular network assembly over time in vivo, intravital imaging was employed using imaging of circulating dextran to identify patent vessels; this imagery also permitted evaluation of blood flow rates as well as localization of differentially labeled input cells (e.g, GFP or dsRed labeled, in the context of blue dextran blood pool imaging).
In order to determine whether the approach of vascular network formation by ASC and EC could be employed with cells that could be derived in a fully autologous approach, implants designed exactly as above, except using endothelial cells obtained from adipose microvasculature, CD 144+ endothelial cells were sorted from fresh preparations of adipose tissue. Further, the behavior of implants incorporating endothelial cells from human umbilical vein wall (HUVECs) were evaluated. Both sources of ECs demonstrated high frequency formation of chimeric RBC-filled vessels in the implants (
Given the secretion of angiogenic factors by ASC, the angiogenic potential of hASC in skeletal muscle was examined in vivo was using two ASC delivery approaches, including: local (intramuscular), and systemic (IV, tail-vein) injections. In the first approach, immunodeficient NOD/SCID mice underwent unilateral femoral artery ligation and received intramuscular injection of either 4×105 human ASCs per hindlimb or media into the m. quadriceps, m. gastrocnemius and m. tibialis anterior of the ischemic hindlimb on the subsequent day (5 injections of 100 μl total). By day 10 of the study (
Based on the known anti-apoptotic effect of HGF, we assessed whether HGF played a critical role in the tissue-preserving effects of ASCs by evaluating the effects of ASC modified either by a lentiviral vector expressing an sh-RNA against HGF (shHGF), or by a control null vector (shCtrl) (
HGF as a key paracrine effector of ASCs.
Explanted islet function are characterized in vitro by the response to glucose exposure, resulting in a burst of cytoplasmic Ca2+, evaluated by ratiometric fura-2
fluorescence imaging; this leads to insulin release into the media, also measurable as a downstream functional index. These assays are illustrated in db/db). (D) Islets were isolated and exposed to 3 mM and then 28 mM glucose for 1 hour.
Islets from normoglycemic C57BL6/J mice were isolated and cultured either alone or on top of an ASC monolayer for 7 days. To assess islet function in both cases, glucose stimulated insulin secretion was measured at the end of experiment. Glucose-stimulated insulin secretion after prolonged culture was significantly higher by islets cultured with ASCs (
Islets from normoglycemic C57BLK6/J mice were isolated and cultured either alone or with an ASC monolayer for 7 days, then stained with green Calcein AM (green) or red Propidium Iodide (PI)(red) to detect live and dead cells, respectively.
To confirm the feasibility of induction of Type I diabetes in immunocompromised (NOD-SCID) mice as a model in which to evaluate islet implant function and stability, these mice were subjected to streptozotocin administration to induce failure of endogenous islets, and monitoring of their glucose levels (
Streptozotocin (STZ) is a nitrosurea compound that is preferentially taken up by the GLUT2 transporter in β cells and acts as an alkylating agent. STZ administered in multiple low doses (MLD-STZ) over 5 days has been shown to reliably induce insulitis and a Type 1 diabetes phenotype within 1-2 weeks via a T cell mediated streptozotocininduced diabetes. NOD
SCID mice were treated in this experiment with STZ at a dose of 55 mg/kg per day for 5 days (
The histology of collagen matrix constructs containing islets admixed with ASC and EC implanted into NOD SCID mice was evaluated. These implants (
STZ, when given as a single high dose, results in β cell ablation and hyperglycemia. Eight week-old NODSCID mice were treated with one injection of STZ at a dose of 150 mg/kg. Two days post-STZ administration, diabetes was documented in treated mice, and either 250 islets alone, 250 islets combined with 2×105 ASCs, or saline carrier (control; no islets) were transplanted under the kidney capsule of recipient mice. Blood glucose tolerance was assessed 16 days post transplantation by challenging with glucose and measuring blood levels over time. Mice co-transplanted with a combination of ASCs and islets exhibited improved glucose tolerance as compared to saline controls (
VEGF secretion by human ASC is markedly repressed by diabetic levels of glucose. The effects of glucose concentration in culture medium on the expression of VEGF by ASC were assessed. ASCs were cultured for seventy-two hours in the presence of physiologic glucose (100 mg/dl, in typical clinical units), or with diabetic or extreme glucose levels (400, and 1000 mg/dl respectively). VEGF secretion into the medium, expressed as ng/106 cells/24 hours was attenuated by more than 50% under hyperglycemic conditions (
To determine the effect of ASC on viability of islet cells, islets from mice were cultured with or without ASC for six days, and then assayed for effect. In the examination of the effect of ASC, live/dead staining was performed and the percentage of live and dead cells were quantitated (green staining indicates live cells and red indicates dead cells). As determined from this analysis, the level of dead cells in the samples with ASC (
To determine whether the ASC introduced into mice migrated to the kidney, a tail vein injection of ASC was conducted followed up by visualization of the ASC through staining. Immunodeficient NOD-SCID mice were first treated with STZ at a level of 40 mg/kg for the first 4 days, as specified to produce type 1 diabetic mouse model. At day 10, GFP-labeled ASC were injected (2×106 ASC) into a fraction of the diabetic mice via the tail vein. On day 13, lung and pancreas samples were harvested from the mice, stained with anti-GFP antibody, and visualized under microscopy (
To determine the effect of ASC on glucose homeostasis in a diabetic mouse model, the same tail STZ induction (45 mg/kg used instead of the previous 40 mg/kg) and tail vein injection of ASC as described in Example 18 was performed, For these mice, a glucose tolerance test was performed (1 g/kg glucose injected intraperitoneally) at 7 days (prior to the ASC administration at day 10), as well as at 7 days and 25 days post injection of the ASC (day 17 and 35 respectively). Serum at pancreata were harvested for analysis at the three time points (Pre-ASC injection, 7 days post injection, and 25 days post injection). IP glucose tolerance test results are shown in
To determine whether ASCs improve beta cell mass in STZ-treated NOD-SCID mice, pancreata were collected 7-10 days post-ASC injection. These collected samples were stained with an insulin-specific antibody to visualize insulin, and beta cell mass was quantitated (
Since the level of Beta cell mass is a balance between cell proliferation and cell death, the level of cell proliferation as affected by ASC was determined. To determine the level of proliferation, an antibody for the proliferation marker Phospho histone 3 (PH3) was used to stain pancreas sections. Under this analysis, all positive nuclei were counted on the islet, but not those outside of the islet. Immunofluorescence visualization of Islets (control or STZ-ASC) stained with anti-insulin, anti-PH3, and DAPI are depicted in
The present international patent application is related to, and claims the priority benefit of, U.S. Provisional Patent Application Ser. No. 61/326,002, filed Apr. 20, 2011, the contents of which are hereby incorporated by reference in their entirety into this disclosure.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/33321 | 4/20/2011 | WO | 00 | 1/7/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61326002 | Apr 2010 | US |
