Patent Application
20120207719
1. Field of the Invention
This invention relates to methods of generating pancreatic islet-like cells, compositions of pancreatic islet-like cells, and methods of using pancreatic islet-like cells.
2. Description of Related Art
Diabetes is a disease characterized by the failure or loss of pancreatic β-cells to generate sufficient levels of the hormone insulin required to meet the body's need to maintain normal nutrient homeostasis. There are two forms of diabetes; type 1 (juvenile) and type 2 (adult late onset). Type 1 diabetes is caused by the complete loss of pancreatic β-cells when the body's own immune system mistakenly attacks and destroys a person's β-cells. For type 2 diabetes the causes are far more complicated and poorly understood, the results of the disease are similar in that the β-cells fail to generate sufficient amounts of insulin to maintain normal homeostasis. The loss of insulin results in an increase in blood glucose levels and eventually leads to the development of premature cardiovascular disease, stroke, and kidney failure. Currently there is no cure for diabetes; however, daily injections of insulin can help regulate blood glucose levels. For these patients, frequent monitoring is important because patients who keep their blood glucose concentrations as close to normal as possible can significantly reduce many of the complications of diabetes, such as retinopathy (a disease of the small blood vessels of the eye that can lead to blindness) and heart disease, both of which tend to develop over time.
More recently, pancreas and islet cell transplantation, have shown some success. Annually, over 1,300 people receive pancreas transplants, with over 80% displaying no diabetic symptoms and are not required to take insulin to maintain their normal blood glucose levels. Pancreas and islet cell transplantation therapies, however, are limited by the availability of donor cadavers. Furthermore, to prevent the body from rejecting the transplanted pancreas or islet cells, patients must take powerful immunosuppressive drugs for the rest of their lives. Immunosuppressive drugs, however, makes patients susceptible to a host of other diseases. Many hospitals will not perform a pancreas transplant unless the patient also needs a kidney transplant because the risk of infection due to immunosuppressant therapy can be a greater health threat than the diabetes itself.
Recently, advances in cell-replacement therapy for diabetes and the shortage of transplantable islet cells have led to an interest in generating a new source of renewable insulin-producing cells, which could be used for transplantation. The progress over the last several years clearly indicates that the stem cell technology may provide the basis for β-cell replacement therapy. Currently, several approaches are being explored to generate insulin-producing cells in vitro, either by genetic engineering of β-cells or utilizing a wide variety of stem or progenitor cells lines. The current stem cell research efforts have been divided between embryonic and tissue specific adult stem cells as potential therapeutic progenitor cells. Recent experiments with embryonic stem (ES) cells have demonstrated that these highly proliferative, pluripotent cells can differentiate into pancreatic-like β-cells. The major problem with ES cells is their pluripotency and the risk that these cells, once transplanted, could induce the formation of tumors. Given that, adult tissue specific stem cells and their progeny have become extremely attractive as a potential cell therapeutic.
Tissue specific stem cells have two distinct advantages over ES cells; first, these cells can be isolated from a mom manageable source such as bone marrow, peripheral blood or other tissues and secondly, they exhibit the capacity to differentiate into a variety of cell lineages under controlled conditions. Stem cell based therapies in which pancreatic insulin-producing cells are generated through controlled differentiation would be beneficial for providing a novel treatment for diabetes. Thus, needs exist in the art to develop a renewable source of human stem cells that can be differentiated from adult stem cells. These adult stem cells should be relatively accessible in order to develop cell types from suitable populations that can be developed in a therapeutic method for production of human pancreatic islet cells. The use of autologous stem cells will provide a therapy for the treatment of diseases and amelioration of symptoms of diabetes.
Provided herein are methods for generating a pancreatic islet-like cell, or monocyte-derived islet cell (MDI). A stem cell may be induced to differentiate into a MDI by contacting the stem cell with at least one differentiation factor. The differentiation factor may be anti-CD40 antibody, epidermal growth factor (EGF), exendin-4, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), insulin-like growth factor-2 (IGF2), LPS, nicotinamide, or combinations thereof. The MDI may express any of the following genes: insulin, IGF2, somatostatin, ngn3, PDX1, islet1, glucose transporter 2 (Glut2), and combinations thereof. The stem cell may express CD117, c-peptide, DPPA5, HES-1, OCT-4, SSEA4, or combinations thereof. The stem cell may be an adult stem cell. The stem cells may be derived from a peripheral blood monocyte. The stern cell may be in a serum-free medium, which may be Megacell DMEM/F12. The stem cell may be isolated from a patient having type 1 or type 2 diabetes.
The MDI may be an α-, γ-, or δ-like cell. A plurality of MDIs may be α-, β-, γ-, or δ-like cells, or a combination thereof. The MDI may secrete insulin in response to an insulin agonist, such as glucose, tolbutamine, and combinations thereof. The MDI may be used to treat a pancreatic-related disorder, such as type 1 diabetes, type 2 diabetes, hyperglycemia, hyperlipidernia, obesity, Metabolic Syndrome, and hypertension.
Also provided herein is a method of treating diabetes, which may comprise administering to a patient in need thereof a MDI.
Provided herein is a method for generating MDIs. The cells may be composed of pancreatic α-, β-, γ-, or δ-like cells or a group thereof. The MDI may be generated by contacting an isolated monocyte-derived stem cell with a differentiation factor. The differentiation factor may be anti-CD40 antibody, EGF, exendin-4, HGF, IGF1, IGF2, lipopolysaccharide (LPS), nicotinamide, or combinations thereof. Exposure to the differentiation factor may cause the stem cell to differentiate into a MDI. The MDIs may be generated or grown in a serum-free media, such as Megacell DMEM/F12. A serum-free medium may be without a serum, such as FBS (fetal bovine serum) or Human AB serum.
The MDI may express β-cell markers such as insulin, c-peptide, islet1, IGF2, ngn3, PDX1, Glut2; or δ-cell markers such as somatostatin, or α-cell markers including but not limited to glucagon. The MDI may secrete insulin in response to glucose, tolbutamine or other insulin agonists or antagonists of insulin and combinations thereof.
a. Stein Cell
The stem cell may be de-differentiated from a monocyte. The monocyte may be derived from human peripheral blood. The monocyte may be de-differentiated by contact with leukocyte inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), or a combination thereof. The de-differentiated stem cell may express stem cell-specific markers, such as CD 117, DPPA5, HES-1, OCT-4, SSEA4, or combinations thereof. In addition, the pancreatic islet-like cluster may secrete a pancreatic factor or hormone including, but not limited to, insulin, c-peptide, glucagon and combinations thereof.
b. Differentiation
The stem cell may be differentiated into a MDI by contact with a differentiation factor or more than one factor in combination. The differentiation factor may be CD40 antibody, EGF, exendin-4, HGF, IGF1, IGF2, LPS, nicotinamide, and combinations thereof. The concentration of CD40 antibody may range from 10 ng/ml to 2 μg/ml. The concentration of EGF may range from 10 ng/ml to 50 ng/ml. The concentration of exendin-4 may range from 10 mM to 40 mM. The concentration of HGF may range from 10 ng/ml to 50 ng/ml. The concentration of IGF1 may range from 10 ng/ml to 50 ng/ml. The concentration of IGF2 may range from 10 ng/ml to 50 ng/ml. The concentration of LPS may range from 10 ng/ml to 100 ng/ml. The concentration of nicotinamide may range from 5 mM to 20 mM. The differentiation factor may be presented to the cells in the presence of culture medium. The culture medium may be LDMEM (low glucose DMEM), HDMEM (high glucose DMEM), DMEM/F12, or Megacell DMEM/F12. The culture medium may be supplemented with serum or serum proteins. Alternatively, the cells may be grown in culture medium without added serum or serum proteins. The differentiation medium may comprise glucose, which may be at a concentration of 2-15 mg/dL or 5-8 mg/dL. The differentiation medium may be changed every three days for optimal differentiation.
Differentiation may be monitored by a variety of methods known in the art. Changes in a parameter between a stem cell and a differentiation factor-treated cell may indicate that the treated cell has differentiated. Microscopy may be used to directly monitor morphology of the cells during differentiation. As an example, the differentiating pancreatic cells may form into aggregates or clusters of cells. The aggregates/clusters may contain as few as 10 cells or as many as several hundred cells. The aggregated cells may be grown in suspension or as attached cells in the pancreatic cultures.
Changes in gene expression may also indicate pancreatic differentiation. Increased expression of pancreatic-specific genes may be monitored at the level of protein by staining with antibodies. Antibodies against insulin, Glut2, Igf2, islet amyloid polypeptide (IAPP), glucagon, neurogenin 3 (ngn3), pancreatic and duodenal homeobox 1 (PDX1), somatostatin, c-peptide, and islet-1 may be used. Cells may be fixed and immunostained using methods well known in the art. For example, a primary antibody may be labeled with a fluorophore or chromophore for direct detection. Alternatively, a primary antibody may be detected with a secondary antibody that is labeled with a fluorophore, or chromophore, or is linked to an enzyme. The fluorophore may be fluorescein, FITC, rhodamine, Texas Red, Cy-3, Cy-5, Cy-5.5. Alexa488, Alexa594, QuantumDot525, QuantumDot565, or QuantumDot653. The enzyme linked to the secondary antibody may be HRP, β-galactosidase, or luciferase. The labeled cell may be examined under a light microscope, a fluorescence microscope, or a confocal microscope. The fluorescence or absorbance of the cell or cell medium may be measured in a fluorometer or spectrophotomer.
Changes in gene expression may also be monitored at the level of messenger RNA (mRNA) using RT-PCR or quantitative real time PCR. RNA may be isolated from cells using methods known in the art, and the desired gene product may be amplified using PCR conditions and parameters well known in the art. Gene products that may be amplified include insulin, insulin-2, Glut2, Igf2, LAPP, glucagon, ngn3, PDX1, somatostatin, ipf1, and islet-1. Changes in the relative levels of gene expression may be determined using standard methods. The expression of α-, β-, γ-, and δ-cell specific markers may show that the MDIs, aggregates or clusters of cells derived from monocyte-derived stem cells (MDSCs) are composed of all four distinct types and three major types of pancreatic cells.
The formation of functional monocyte-derived islets (MDIs) may be determined by monitoring the synthesis and secretion of factors such as insulin and c-peptide during the differentiation of MDSC-derived MDIs. Contact with high levels of glucose may stimulate the MDIs to secrete insulin or c-peptide. Contact with tolbutamide or other insulin agonists may stimulate the MDIs to secrete increased levels of insulin. The levels of insulin or c-peptide may be measured in the culture medium of the different cells the using an ELISA protocol. Other methods known in the art may be used to monitor the secretion of insulin or c-peptide by the differentiated cells.
c. Proliferation
The MDI may be induced to proliferate by contacting it with differentiation medium comprising glucose, which may be at a concentration of 5-40 mg/dL, 10-25 mg/dL, or 18-25 mg/dL. The proliferation may be monitored by staining the MDI with propidium iodide or Ki-67, which may be followed by flow cytometry.
The MDI may be used to replenish a cell population that has been reduced or eradicated by a disease or disorder, as a treatment for such a disease or disorder, or to replace damaged or missing cells or tissue(s). The MDI may be given autologously or to a allogenically compatible subject.
Diabetes mellitus is an example of a disease state associated with an insufficiency or effective absence of certain types of cells in the body. In this disease, pancreatic islet β-cells are missing or deficient or defective. The condition can be treated, or at least one of its symptoms ameliorated, by insertion of MDIs. The MDIs may be derived from MDSC isolated from a patient that is healthy, or who may have type 1 or type 2 diabetes. Both type 1 diabetes mellitus (juvenile-onset diabetes or insulin-dependent diabetes mellitus) and type 2 diabetes mellitus (adult-onset diabetes) may be treated with MDIs. Other disorders that may be treated with MDIs include hyperglycemia, hyperlipidemia, obesity, Metabolic Syndrome, and hypertension.
MDIs may be inserted into the body by implantation, transplantation, or injection of cells. The cells may be introduced as single cells or clusters of cells. Methods of transplanting pancreatic cells are well known in the art. See for example, U.S. Pat. Nos. (4,997,443 and 4,902,295) that describe a transplantable artificial tissue matrix structure containing viable cells, preferably pancreatic islet cells, suitable for insertion into a human. Moreover, since MDIs may be derived from peripheral blood monocytes of the same individual who will later receive the cell transplantation, the use of immunosuppressive agents may not be necessary.
Also provided herein are compositions comprising the MDIs. The compositions may include a single cell, an aggregate of cells. or a tissue-like cluster of cells. The composition may comprise 10-10,000, 10-1000, or 10-1000 MDIs. The composition may also comprise 5-60% α-cells, 30-95% β-cells, 1-30% δ-cells. 0-5% γ-cells, or combinations thereof.
As various changes could be made in the above compounds, methods, and products without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense,
The following examples illustrate, but do not limit, the invention.
Isolated peripheral blood monocytes were plated in a 2:1 mixture of Megacell DMEM/F12 medium (Cat. No. M4192, Sigma-Aldrich) and AIM V medium (Invitrogen) and cultured overnight at 37° C. and 5% CO2. The culture medium was supplemented with 4 mM L-glutamine and penicillin-streptomyocin. The cells were plated on FALCON vacuum-gas plasma treated plates. After 24 hours, the culture medium was removed and the cells were gently washed three times with 1× HBSS containing 2 mM EDTA. De-differentiation medium, which was Megacell DMEM/F12 or LDMEM (low glucose DMEM) or HDMEM (high glucose DMEM) containing 10 ng/ml leukocyte inhibitory factor (LIF; Cat. No. LIF1010, Chemicon) and 25 ng/ml macrophage colony-stimulating factor (M-CSF: Cat. No. GF053, Chemicon), was added. After three days, the medium was removed and replaced with fresh de-differentiation medium. After 6 days in culture the cells had de-differentiated into monocyte-derived stem cells (MDSCs).
MDSCs were washed two times with 1× HBSS. Pancreatic differentiation medium was added to the cells and they were cultured. Pancreatic differentiation medium comprised Megacell DMEM/F12 (or LDMEM or HDMEM) supplemented with L-glutamine, penicillin, and streptomyocin, as well as 1 μg/ml CD40 antibody (R&D Systems; catalog number MAB6321, clone 82111), 100 ng/ml LPS (Chemicon; catalog number LPS25), 1× ITS, 10 mM nicotinaminde, 1% N2 supplement, 25 ng/ml EGF (Chemicon; catalog number GF001), 20 ng/ml HGF (Chemicon; catalog number GF116), 25 ng/ml IGF1 (Chemicon; catalog number GF006), 25 ng/ml IGF2 (Chemicon; catalog number GF007), and 20 mM Exendin-4 (Sigma-Aldrich; catalog number E7144).
Aggregates of cells were observed after 18 hours in pancreatic differentiation medium (
After 6 days in culture, the aggregates detached from the plates and were free floating clusters. Beginning at 4-6 days, pancreatic factors or hormones such as insulin, c-peptide and glut2 were initially detected in MDIs derived from MDSCs that were cultured under pancreatic differentiation conditions, while no pancreatic factors or hormones were detected in de-differentiated MDSC cultures. At this time, the cells were challenged with high glucose conditions. For these experiments, cells were exposed to pancreatic differentiation medium containing 25 mM glucose (normal pancreatic differentiation medium contained 5 mM glucose). The number and size of the aggregates or clusters increased in the presence of high glucose conditions. In addition, the expression of several genes was also changed (see Example 2). Cultures were shown to maintain their growth over a month by changing the pancreatic differentiation medium containing 25 mM glucose every three days.
To monitor the differentiation of MDSCs into MDIs, the expression of pancreatic-specific genes was analyzed by real time PCR. The following cell-specific markers were examined: β-cell specific markers were Glut2, IAPP, Igf2, insulin, ngn3, and PDX1; α-cell specific marker, glucagon; and δ-cell specific marker, somatostatin. MDSCs were generated as described in Example 1. One set of MDSCs was maintained in de-differentiation medium. The second set was cultured in pancreatic differentiation medium for six days and then challenged with high glucose conditions.
For each time point, cells were collected (1×105 to 3×106 cells/well) and RNA was isolated using Qiagen Rneasy Kit (Cat. No. 74103) following the manufacturer's instructions. First strand cDNA was synthesized by mixing 1 ng-5 μg of RNA with 1 of 500 μg/ml of oligo(dT) (Invitrogen; catalog number 55063), 1 μl of 10 mM dNTPs (Invitrogen; catalog number 18427-013), and water to equal 12 μl. The mixture was heated to 65° C. for 5 minutes and the chilled on ice. Then 4 μl of 5× First-strand buffer, 1 μl of 0.1 M DTT (Invitrogen; catalog number 18427-013), 40 units of RNaseOUT (Invitrogen: catalog number 10777-019), and 200 units of Superscript III RNaseH− RT (Invitrogen; catalog number 18080-093) were added. The tube was gently mixed and incubated at 50° C. for 60 minutes. The tube was spun and the enzymes were inactivated by heating to 70° C. for)5 minutes. The concentration of cDNA was estimated using a spectrophotometer.
For real time (quantitative) PCR, 100 ng of cDNA was mixed with 200 nM of each primer, and 0.5 volume of SYBR green qPCR SuperMix-UDG with ROX (Invitrogen; catalog number 11744). The cycling parameters were 50° C. for 2 minutes, 95° C. for minutes, followed by 40 cycles of 60° C. for 30 seconds and 95° C. for 30 seconds. Primers were designed by Primer3 software with TM=60° C. See Table 1 for primer sequences and sizes. All PCR reactions were run in duplicate and averaged based on ΔCT values. To determine the relative gene expression, the ΔCT values for controls (GADPH and β-actin) were compared to pancreatic gene expression. To calculate the percent of relative expression, the following formula was used:
R.E. (relative expression )=2n−(ΔCT gene−ΔCT GAPDH)×100
To assess the functionality of the differentiated MDIs, insulin secretion was measured under the different conditions using an ELISA kit (Diagnostic Systems Labs Inc; Cat. No. DSL-10-1600). For this “one-step” sandwich-type Immunoassay, standards, controls, and unknown serum samples were incubated with an HRP-labeled anti-insulin antibody in microtitration wells that had been coated with another anti-insulin antibody. After incubation and washing, the wells were incubated with the substrate tetramethylbenzidine (TMB). An acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 and 620 nm. The absorbance measured was directly proportional to the concentration of insulin present. A set of insulin standards was used to plot a standard curve of absorbance versus insulin concentration from which the concentration of insulin in the unknown samples was calculated.
After 24 hours of high glucose challenge the pancreatic aggregates synthesized 28.8 μl U/ml of active insulin into the medium (
To further analyze the function of the aggregates of MDIs, an ELISA kit (Diagnostic Systems Labs Inc; Cat. No. DSL-10-7000) was utilized to measure the level of c-peptide secreted by the cells. In this assay, standards, controls and unknown serum samples were incubated with an HRP-labeled anti-c-peptide antibody in microtitration wells that had been coated with another anti-c-peptide antibody. After incubation and washing, the wells were incubated with the substrate tetramethylbenzidine (TMB), An acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 and 620 nm. The absorbance measured was directly proportional to the concentration of C-peptide present. A set of c-peptide standards was used to plot a standard curve of absorbance versus c-peptide concentration from which the concentration of c-peptide in the unknown samples was calculated.
To further examine the function of these differentiated islet-like cells, the effects of tolbutamide and glucose were studied in parallel. Cells were exposed to increasing concentrations of glucose (5, 6, 7, 10, 12, 15, 18, 21 and 25 mM) in the presence or absence of 10 μM tolbutamide for periods of 12 minutes each. The secretion of insulin was analyzed using an insulin ELISA kit (see Example 3). For these experiments pancreatic clusters were collected and plated in a 24 well format with 2 ml of Krebs-ringer bicarbonate buffer containing 5 mM glucose.
The following demonstrates that monocyte-derived islet cells (MDIs) exhibit increased proliferation in response to pancreatic medium and high glucose levels (25 mM). To assay the proliferation of MDSCs and MDIs the expression of Ki-67, a marker strictly associated with cell proliferation, was assayed. During interphase, this antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population.
The effects of high glucose on MDI proliferation as measured by Ki-67 is shown in
This effect is further illustrated in Table 2 below, which shows the percentage of cells in S. G0/G1, and, G2/M phases at days 2, 6, 8, 12, and 17 as measured by propidium iodide (PPI) levels in flow cytometry analysis. Higher rates of proliferation were indicated by the higher percentage of cells in S phase.
The above results indicate that pancreatic medium with high levels of glucose increases MDI proliferation.
The following demonstrates that high glucose levels increase the number of MDI. aggregates. MDSCs were cultured in serum free conditions in DMEM/F12 medium for 6 days, and then cultured in pancreatic medium containing 5 mM glucose. Pancreatic aggregates formed into small free floating clusters after 3 days in pancreatic medium. In low glucose conditions (5 mM), the cultures generated approximately 200 clusters per well in a 6 well plate (Falcon). However, when MDIs were cultured in high glucose (25 mM), approximately 600 clusters were generated per well in a 6 well plate. For these studies 20×106 PBMCs per well were plated.
The following demonstrates that high glucose levels increase MDI cluster size. MDSCs were cultured in serum free conditions DMEM/F12 medium containing LIF and M-CSF for 6 days for the initial de-differentiation. After 6 days, MDSCs were treated with pancreatic medium containing 5 mM glucose. During this period, pancreatic aggregate formation was observed. Continued treatment of cells with pancreatic medium with low glucose eventually produced free floating clusters. After 6 days in low glucose pancreatic medium, MDIs were treated with low- or high-glucose (5 mM or 25 mM, respectively). Under these conditions increases in both size and number of MDIs in culture were observed. The results of these experiments is shown in
Table 3 shows the size of the MDIs in microns using a Leica DMire2 microscope with 5.1 scope imaging software. Multiple samples were measured from 6 different MDI cultures and the mean value of the size was calculated and plotted.
The above results indicate that high levels of glucose in pancreatic medium increase MDI size and number.
The following demonstrates that MDIs derived from MDSCs using pancreatic medium with high glucose levels express endocrine-specific markers in association with increased rates of proliferation. For these experiments, expression of endocrine-specific markers was examined by immunofluorecence using antibodies specific for β-cells, including insulin, c-peptide, and Pdx1, and for α-cells (glucagon). The expression profiles of these factors in MDIs were observed at various stages.
The results above demonstrate that MDIs express endocrine specific markers and are composed of the major pancreatic cell types (α, β and δ). Real time PCR showed that ngn3, a known marker for the pancreatic progenitors known as the γ-cells or PP cells, was expressed. The composition of the MDIs was approximately >60% β-cells, 10-25% α-cells, and 1-5% δ-cells. MDI exhibited a similar cellular composition to that observed in human pancreatic islets.
Furthermore, MDIs have an increased rate of proliferation when cultured in high glucose conditions. This increased proliferation correlates with an increased expression of ngn3, pdx1 and somatostatin biomarkers for the formation of new islet progenitors within the MDIs cultures.
The following demonstrates that MDIs can be derived from MDSCs of diabetic subjects. To test the ability to generate both MDSCs and MDIs from both type 1 and 2 diabetic subjects, peripheral blood monocytes (PBMCs) were isolated from subjects with diabetes and MDSCs were produced using de-differentiation medium. To determine if functional MDIs can be generated from MDSCs derived from subjects with diabetes, their MDSCs were cultured under pancreatic differentiation conditions.
PBMCs were isolated from 14 subjects with diabetes. These subjects were diagnosed with insulin-dependent type 1 or type 2 diabetes. Multiple blood draws were performed on each of these subjects, and each draw was separated by at least 2 weeks. This provided duplicate samples to ensure reproducibility.
MDSCs were isolated and generated using methods as described above for deriving pancreatic islets, and were monitored for up to 30 days in culture. To monitor c-peptide c-peptide ELISA (DSL) and Western blot analysis were performed. Immunohistochemical and PCR analyses were performed on samples to examine the expression of several pancreatic and proliferative markers during the course of the islet formation. Luminex was used to examine the levels of insulin, c-peptide and glucagon in each subjects' plasma.
The results of generating MDSCs and MDIs from subjects with diabetes is summarized in Table 4 below.
Additionally, levels of insulin, glucagon, and glp-1 in plasma collected from diabetic subjects were measure by performing a Luminex assay (Linco) according to the manufacturer's protocol. This provided baseline levels for these specific hormones. 25 μL of plasma was used for each assay and all samples were run in duplicate to provide more accurate and reliable data.
6.2 pM
6.2 pM
The results above demonstrate that MDIs can be formed from MDSCs isolated from subjects with diabetes.
The following demonstrates that MDIs generated from MDSCs isolated from subjects with type 1 or type 2 diabetes express α- and β-cell markers. To examine the functionality of MDIs generated from subjects with type 1 and 2 diabetes, immunofluorescene staining with specific antibodies for β-cell markers (c-peptide and Pdx1) and the α-cell marker (glucagon) was performed.
In addition to expressing α- and β-cell markers, MDIs derived from subjects with diabetes secrete insulin. This was demonstrated by performing ELISA and Luminex assays on both plasma collected from subjects' blood and on the supernatant collected during MDI growth. ELISA assays were performed using either DSL or Mecodia kits following standard operating procedures. Luminex was performed using a Linco diabetes kit containing insulin, c-peptide and glucagon. Each sample was run in triplicate and analyzed against blank and standard controls.
Table 6 also shows insulin secretion by MDIs derived from subjects with type I diabetes. An ELISA insulin kit (DSL) was used to measure the amount of insulin secreted by MDIs between days 15 and 40. The level of insulin in the subjects' plasma at the time of collection was also examined.
The above results demonstrate that MDSCs and MDIs can be generated from subjects with type 1 (n=7) or type 2 (n=7) diabetes. These MDIs express endocrine-specific markers and are able to synthesize and secrete insulin and c-peptide. ELISA and Luminex analysis demonstrated the ability of MDIs from subjects with diabetes to synthesize and secrete insulin and c-peptide in a glucose-responsive manner.
The following demonstrates that MDIs derived from MDSCs isolated from human subjects are capable of treating diabetes in mice. To examine the ability of insulin-producing cells generated in vivo to reverse hyperglycemia, a streptozotocin (STZ)-induced diabetes NOD/SCID mouse model was used.
Hyperglycemia was induced in 8-10 week-old male NOD/SCID mice (Taconic laboratory) by 3 injections of 40 mg/kg of body weight streptozotocin (STZ) that had been freshly dissolved in 0.1 M citrate buffer. Stable hyperglycemia developed between 3-5 days after STZ injections, resulting in blood glucose levels between 300 to 600 mg/dL. Glucose levels in tail vein blood were measured using a glucometer. The animals were grafted with cells or buffer vehicle 48 hours after establishing stable hyperglycemia.
Mice were transplanted with approximately 500 insulin producing clusters (or approximately 1×106 cells in suspension) or 5×106 MDSCs derived from human subjects into the right subcapsular renal space. Blood glucose was then monitored every 2 days for 6-12 weeks after the transplantation. The transplants were excised by unilateral nephrectomy to test for euglycemia reversal, and glucose monitoring was continued. At the end of the experiment, serum was taken from the mice for insulin and c-peptide analysis. Insulin and c-peptide levels were monitored using ELISA and Luminex assays. Concurrent studies were performed on groups of 20 to 40 mice.
Groups A-D were treated as described below (total of 24 mice):
(A) Transplanted mature MDSCs and monitored for 3-12 weeks, transplants were excised, followed by continued glucose monitoring for 2 additional weeks.
(B) Transplanted 500 islet clusters—early-(cultured under high glucose conditions for 3-6 days)(i.e., MDIs at day 15, or “d15”) and monitored for 3-12 weeks, transplants were excised, followed by continued glucose monitoring for 2 additional weeks. The d15 MDIs had been exposed to high glucose conditions for 3 days and exhibited an increase in the expression of PDX1, somatostatin and ngn3. The d15 MDIs also expressed a low level of insulin. The clusters also had an increased rate of proliferation. The size of the d 15 MDIs was 100 to 300 microns. In addition the total number of d15 MDIs in a well of a 6 well plate was 100 to 500 clusters.
(C) Transplanted 500 islet clusters—late-(cultured under high glucose conditions for 7-12 days)(i.e., MDIs at d23) and monitored for 3-12 weeks, transplants were excised, followed by continued glucose monitoring for 2 additional weeks. The d23 MDIs had been exposed to high glucose for 11 days and exhibited a increased level of insulin (2-8 ng/ml) per well of 6 well plate. By immunofluorescene the d23 MDIs exhibited expression of insulin, glucagon and somatostatin within the clusters. The proliferation rate of d23 MDIs was relatively unchanged compared to d15 MDIs. The size of the d23 MDIs was 200-1000 microns. The total number of d23 MDIs in a well of a 6 well plate was 200-1000 clusters.
(D) Sham transplant of krebs-ringer bicarbonate buffer saline without Ca2 (Vehicle control) injection monitored for 6 weeks, transplants were excised, followed by continued glucose monitoring for 2 additional weeks
MDSCs were generated from buffy coats obtained from a Regional Blood Bank from healthy human donors following standard operating procedures. These samples were screened by the blood center prior to shipment. The samples were processed via a common lymphocyte separation method in which the mononuclear fraction was collected, washed and counted using a Vi-cell particle counter as previously described. MDSCs were prepared from PBMCs as described above.
PBMCs collected from the mononuclear fractions were then resuspended in medium and seeded onto treated tissue culture dishes. The cells were then incubated at 37° C. in 5% CO2. When MDSCs were fully developed, a subset was harvested and prepared for control injections.
To generate MDIs, MDSCs were further grown in de-differentiation medium for 6 days. MDSCs were then washed and fed with a pancreatic medium containing low glucose (5 mM) for 6 days. Next, cultures were treated with pancreatic medium containing high glucose (25 mM). MDIs were then incubated at 37° C. in 5% CO, for a either 3 days or 11 days before harvesting. MDIs were harvested by placing them in a falcon tube, followed by centrifugation at 500 rpm for 5 minutes. The medium was then removed and replaced with pancreatic medium. Cells were stored at 37° C. until injection.
Prior to injection into NOD/SCID mice, MDIs were centrifuged at 500 rpm for 5 minutes and washed in fresh pancreatic medium. The cells were then centrifuged again as described above and resuspended in 50 μl pancreatic medium. Next, the cells were collected into a small gauge needle and injected through the kidney into the kidney capsule. All mouse surgeries were performed following approved animal protocols under sterile conditions.
Prior to injection into mouse kidney capsules, MDSCs and islet-like clusters were characterized by flow cytometry, immunohistochemistry and Real Time PCR. The phenotype of MDSCs was determined by using endocrine-specific markers which included insulin, c-peptide, somatostatin and glucagon. To test the functionality of MDIs, the expression of insulin, c-peptide, glucagon, and somatostatin were examined both by immunohistochemistry and Real Time PCR.
For PCR-based characterization, total RNA was extracted from both MDSCs and MDIs, and cDNA synthesized using standard protocols. To determine the relative expression of several pancreatic genes, Sybr green and/or Taqman Real Time PCR assays were used. All samples are compared to GADPH and B-actin standards to determine the relative gene expression.
Following injection of MDSC control cells, saline control, or d15 or d23 MDIs into STZ-induced hyperglycemic NOD/SCID mouse kidney capsules, blood glucose levels were monitored over 60 days. The ability of early MDIs (d15) were compared to late MDIs (d23) in lowering blood glucose levels.
Table 7 also shows the results of measuring blood glucose levels in wildtype and STZ-induced NOD/SCID mice injected with saline control, MDSCs, or d15 or d23 MDIs.
Body weights of STZ-induced hyperglycemic NOD/SCID mice transplanted with day 15 MDIs were also examined for 73 days, and compared to wildtype, and STZ-induced hyperglycemic NOD/SCID mice injected with either saline control. MDSCs, or d15 MDIs. The results of these experiments are shown in
Z induced
indicates data missing or illegible when filed
α-(glucagon) and β-cell (insulin) marker expression was also examined n STZ-induced hyperglycemic NOD/SCID mice transplanted with day 15 MDIs. Kidneys from NOD/SCID mice injected with d15 MDIs were collected within an hour of injection and fixed in 10% formalin overnight, and then processed in paraplast. Tissues were then sectioned and stained with antibodies for insulin and glucagon.
Expression of α- and β-cell markers were also analyzed in plasma from the above-described NOD/SCID mice. Plasma was collected from untreated control mice, and from STZ-induced hyperglycemic NOD/SCID mice that were injected with MDSCs or MDIs. The results of these experiments is shown in Table 9. An increase in the level of human glucagon in mice #134 and #145 was observed. Both mice were injected with MDIs. Mouse #134 had been injected with early islets and #145 with late islets. Both mice exhibited a decrease in blood glucose levels. No change in glucagon levels were observed for untreated or MDSC-injected mice.
6.2 pM
6.2 pM
The experiments described above demonstrate that MDSCs have no effect on blood glucose levels. Furthermore, d15 MDIs injected into STZ-induced NOD/SCID hyperglycemic mice are capable of reducing blood glucose levels to near-normal levels for a prolonged period of time, and restoring body weight to normal range. d23 MDIs also lower blood glucose levels to 300 mg/dL compared to levels of over 500 mg/dL in STZ-induced mice. However d23 MDIs were only effective for 6 weeks, after which mice returned to a diabetic state.
The above experiments are consistent with early (d15) MDIs being capable of proliferating or renewal within the kidney capsule, although the more terminally differentiated late (d23) MDIs have limited proliferation. In addition, an increase in the secretion of human glucagon was observed in STZ-induced NOD/SCID mice that were injected with MDIs, and these mice had lower blood glucose levels. The level of glucagon detected in NOD/SCID mice transplanted with MDIs was within human physiological ranges.
The results described above demonstrate that MDIs generated from human MDSCs are capable of treating symptoms of diabetes, including elevated blood glucose levels.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/US07/68303 | May 2007 | US | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12299590 | Nov 2008 | US |
| Child | 13428651 | US |
