KR101452877B1 - Method for Preparing Cell Implant comprising Early-Endothelial Progenitor Cells and Islet Cells - Google Patents

Abstract

The present invention relates to a method for preparing a cell implant which comprises: (a) a step for cultivating early-endothelial progenitor cells of a mammal; (b) a step for cultivating islet cells of a mammal; and (c) a step for preparing a cell implant by mixing the cultivated early-endothelial progenitor cells and islet cells. According to the present invention, the cell implant of the present invention provides excellent ability to reach normal blood sugar and ability to create blood vessels.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of preparing a cell-plant containing an early-endothelial progenitor cell and an islet cell,

The present invention relates to a method for producing a cell api comprising an early-endothelial progenitor cell and an islet cell.

With the establishment of the islet transplantation protocol in Edmonton, Canada, clinical islet cell transplantation has emerged as a possible treatment for patients with type 1 diabetes (1). However, low engraftment of transplanted islet cells is a major factor in long-term failure to control blood glucose (2). Although islet cells should be successfully transplanted through new blood vessel regeneration and blood flow control within a few days after transplantation, transplanted somatic cells are exposed to lower vascular density, oxygen partial pressure and nutrients compared to endogenous islet cells, Cells undergo the process of death, resulting in difficulties in the normal engraftment of insulin cells and control of the secretion of insulin. The completion of the new vascular network takes place approximately 10-14 days after implantation (3,4).

Bone Marrow-derived Stem Cells have been recognized as important therapeutic agents for clinical cell therapy. Bone marrow-derived stem cells include various cells, such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs). Bone marrow-derived EPCs play an important role in the regeneration of injured ischemic organs and contribute to the induction of neovascularization in patients with ischemic disease (5-7).

EPC reports that there are at least two types during the incubation period under laboratory conditions. EPCs or endothelial colony-forming units (CFU-FCs) and late-EPCs (endothelial colony forming cells) (ECFs) . Both types of EPCs differ in shape, culture period, cell proliferative capacity, and expressed protein, but have endothelial cell functional properties and have been reported to improve on angiogenesis in animal models (8). Although late-vascular endothelial progenitor cells have the ability to repair vascular damage, previous clinical results have shown that, considering the effective timing of bone marrow-derived cell delivery after myocardial infarction is 4-10 days, Because of the need for the expansion of late-vascular endothelial progenitor cells, the clinical approach lacks practical access to autologous or allogeneic cell therapy (11). In addition, the origin of late-vascular endothelial progenitor cells still can not be defined for clinical applications, and clinical trials have yet to be conducted. This has the disadvantage of unexpected risk of cancer development in long-term laboratory culture conditions such as mesenchymal stem cells (MSCs) (12,13).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to overcome problems such as low engraftment rate of transplanted islet cells of conventional Islet Cells transplants. As a result, it was found that the cells obtained by culturing the early-endothelial progenitor cells and the pancreatic islets before and after the transplantation exhibited excellent normal glucose uptake ability and vascularity, .

Accordingly, an object of the present invention is to provide a method for producing a plant cell.

Another object of the present invention is to provide a cell plant.

It is another object of the present invention to provide a pharmaceutical composition for improving, preventing or treating diabetes mellitus.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a plant cell for an islet cell transplantation comprising the steps of:

(a) culturing mammalian early-endothelial progenitor cells;

(b) culturing mammalian islet cells (Islet Cells); And

(c) mixing the cultured pre-endothelial progenitor cells and the islet cells to prepare a cell plant.

The present inventors have sought to overcome problems such as low engraftment rate of transplanted islet cells of conventional Islet Cells transplants. As a result, it was confirmed that the cell plants produced by culturing the early-endothelial progenitor cells and the pancreatic islets showed excellent engraftment and vascularity.

In order to prepare the cell plants of the present invention for pancreatic cell transplantation, mammalian pre-endothelial progenitor cells are cultured. According to one embodiment of the present invention, the early-endothelial progenitor cells used in the present invention are isolated early-endothelial progenitor cells. According to one embodiment, the early-endothelial progenitor cells used in the present invention are bone marrow-derived early-endothelial progenitor cells.

Culturing of early-endothelial progenitor cells can be carried out in a variety of media known in the art, such as EBM-2 (endothelial basal medium-2) supplemented with EGM-2 SingleQuots and FBS (fetal bovine serum).

The mammal of the invention is a human, a bovine, a horse, a pig, a goat, a dog, a cat, a chicken, a mouse, a rat, a rabbit or a guinea pig. According to one embodiment of the invention, the mammal is a human or a pig.

The pre-endothelial progenitor cells for producing the cell apiary plant of the present invention are precursor cells of the pre-stage before differentiation into endothelial cells, which means cells capable of differentiating into endothelial cells under specific conditions. According to one embodiment of the present invention, the pre-endothelial progenitor cells are obtained by separating bone marrow from the thighs and tibia of a mammal to remove erythrocytes and culturing the obtained mononuclear cells in an endothelial cell differentiation medium for 5 to 12 days it means.

The pre-endothelial progenitor cells of the present invention have a spindle-shaped morphology. According to one embodiment of the present invention, the pre-endothelial progenitor cells exhibit a fusiform shape when cultured in an endothelial cell differentiation culture medium for 7-10 days.

The pre-endothelial progenitor cells of the present invention have characteristics of endothelial progenitor cells, mononuclear cells, and hematopoietic stem cells. According to one embodiment of the present invention, the pre-endothelial progenitor cells of the present invention are selected from CD106, CD31, Flk-1, eNOS, vWF and VE-cadherin, markers of endothelial progenitor cells, CD11b and CD45, Express CD34 and Sca-1, markers of hematopoietic stem cells.

As used herein, the term " expression " refers to gene expression analysis methods commonly used in the art, such as real-time RT-PCR (see Sambrook, J. et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Western blot ("Humira Press. Retrieved 2010"), FACS ("Invention Systems for Westerns: Chemiluminescence vs. Infrared Detection, 2009, Methods in Molecular Biology, Protein Blotting and Detection, Flow cytometry or Fluorescence-activated cell sorting, Loken MR (1990). Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd ed.) Wiley, pp. 34153) Immunocytochemistry Methods and Protocols. Edited by Lorette C. Javois, When analysis of gene expression or protein expression is analyzed according to molecular biological experiments such as the 2nd edition, 1999. Human Press, etc., it is analyzed that gene expression and / or protein expression is higher than that of the control plant without transplanting the cell plant of the present invention .

Early in the present invention - endothelial progenitor cells DiⅠ-ac-LDL (Acetylated Low Density Lipoprotein labeled with 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindo-carbocyanine perchlorate) absorption capacity and BS-1 (Bandeiraea simplicifolia ) lectin binding ability. The DiI-ac-LDL uptake is a marker for identifying endothelial cells. When the cells are contacted with DiI-ac-LDL, the lipid protein is degraded by lysosomal enzymes and the fluorescent dye, DiI, accumulates in the intracellular membrane Can be visually confirmed. The BS-1 lectin binding ability is a marker for confirming endothelial cells. When BS-1 lectin is reacted with endothelial cells, endothelial cells show binding ability to BS-1.

One of the main features of the present invention is that the pre-endothelial progenitor cells and the pancreatic islets are individually cultured and then mixed to prepare a cell plant for pancreatic islet transplantation.

According to one embodiment of the invention, the pre-endothelial progenitor cells of the invention are cultured for 5-12 days. According to another embodiment of the present invention, the pre-endothelial progenitor cells of the present invention are cultured for 7-10 days.

On the day before the preparation of the cell plant for the pancreatic cell transplantation of the present invention, the cultivation of the pancreatic cell is initiated.

According to one embodiment of the present invention, the pancreatic islet cells are cultured for 16-24 hours.

Another of the main features of the present invention is that the cell plant mixes the premature endothelial progenitor cells and the pancreatic islet cell immediately before transplantation.

According to one embodiment of the present invention, the plant has a cell number mixing ratio of 1000: 1 to 10000: 1. According to another embodiment of the present invention, the cell plant has a cell number mixing ratio of 2500: 1 to 7500: 1.

The cell plant of the present invention has excellent ability to reach normal blood glucose.

According to an embodiment of the present invention, the plant cell has a normal glucose uptake capacity of 1.5 to 3.0 times as much as an islet cell of an islet cell.

As used herein, the term " normal glucose uptake ability " refers to the efficacy of a cell plant to transplant a cell plant based on a mouse and to reach a blood glucose level of less than 11.1 mmol / L when 1 g / kg of glucose is injected do. More specifically, when a cumulative diabetes reversal curves is made for a cell plant, the ability to reach normal blood glucose levels as a percentage of the recipient reaching a blood glucose level of less than 11.1 mmol / L (see Figure 2g) .

According to another embodiment of the present invention, when the cell plant of the present invention is transplanted, it exhibits a normal glucose uptake ability of about 82%, and the islet cell alone has about 38% .

The cell plant of the present invention has excellent vascularity.

According to one embodiment of the present invention, the plant cell has an angiogenesis ability of 2 to 3 times as much as an islet cell of an islet cell.

As used herein, the term " angiogenesis " means an angiogenic potential of a cell phyla. Specifically, in order to confirm the degree of angiogenesis by a cell phyla, a CD31 antibody (based on a mouse) (See Figs. 4E and 4F).

According to another embodiment of the present invention, when the cell plant of the present invention is transplanted, the islet cell alone exhibits an angiogenic number increased by 2 to 3 times as compared with the plant.

According to another aspect of the present invention, the present invention provides a cell plant for pancreatic cell transplantation produced by the above method.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for improving, preventing or treating diabetes comprising a cell plant produced by the above method of the present invention.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered parenterally, and in the case of parenteral administration, it can be administered by local transplantation or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Preferably, the dosage of the pharmaceutical composition of the present invention is 1-10000 cells / kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents

Since the composition for improving, preventing, or treating diabetes mellitus and diabetes of the present invention is produced by the method for producing a cell mugwort, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for preparing a cell plant for insect cell transplantation, a cell plant, and a composition for preventing or treating diabetes.

(b) The cell plant of the present invention provides excellent normal blood glucose accessivity and vascularity.

(c) The cell plant of the present invention can overcome the low incidence of conventional islet cell transplantation.

FIGS. 1A-1E illustrate the features of early-endothelial progenitor cells (EPCs) from mouse bone marrow-derived. 1A shows a microscope image of an early-EPC cultured in an endothelial cell culture medium for 7 days. The scale bar represents 100 mu m. FIG. 1B shows the results of flow cytometry analysis of CD31, Flk-1, CD106, CD34, CD117, Sca-1, CD11b and CD45 expressed on the surface of the early-EPC. FIG. 1C shows the results of confirming Di-ac-LDL absorption ability and BS-1 lectin binding ability of the early-EPC on the 7th day of culture. The scale bar represents 50 mu m. FIG. 1D shows the CD31 of early-EPC. Flk-1, vWF and eNOS expression were confirmed by RT-PCR. Figure 1e shows the results of immunochemical staining of endothelial cell markers CD31, VE-cadherin and Flk-1.
Figs. 2A to 2G show blood glucose levels in an unabbreviated state after transplantation. FIGS. 2A to 2C show the percentage of islet cells reached to normal blood glucose according to the number of transplanted islet cells. FIG. 2 (a) shows the islet cell transplantation group (0/7), FIG. 2 (b) shows the transplantation group of 200 transplanted islet cells (6/12) . FIGS. 2d to 2f show changes in blood glucose when islet cells alone (n = 13), islet cells + non-EPC (n = 14) and islet cells + BM-EPC were transplanted (n = 17). Figure 2g shows cumulative diabetes reversal curves (percent of mice reaching normal blood glucose levels with a blood glucose concentration of less than 11.1 mmol / l) reaching normal blood glucose over time (* p < 0.05, vs. Islet cell alone or pancreatic islet cell + non-EPC).
Figures 3A-3C show insulin measurements from sera in the glucose tolerance test and fasted state on day 28 after transplantation. Figure 3a shows glucose tolerance test for islet cell + EPCs cavity graft (white squares) and islet cell single graft (black circle). Figure 3b shows AUC _glu (area under the glucose curve) for glucose _tolerance test. FIG. 3c shows serum insulin levels measured at 0 min and 30 min after glucose was injected in a fasted state (*: p <0.05, **: p <0.01).
FIGS. 4A to 4F show that when the islet cell and the early-EPC were transplanted simultaneously, the angiogenesis was improved in the area where the islets were transplanted. 4A and 4B show that the islet cells isolated from the genetically modified mouse (GFP-Tg) are consistent with those stained with insulin antibody, and the right panel of FIG. FIG. 4B shows a comparison of the area of the area where the islet cell was fixed between the islet-only transplant group and the transplant group of the islet cell + early-EPC. Figures 4c and 4d show the shape and composition of the established islet cells and quantitatively show the number of glucagon. The white dotted rectangle is an enlarged photograph to show the composition of each group of glucagon, and the right panel of FIG. 4C shows the distribution of glucagon in one islet cell of the mouse (rod bar size is 50 μm). Figures 4e and 4f show the degree of angiogenesis in the fixed area and the staining of blood vessels quantitatively shows the number of blood vessels using CD31 antibody (red) (Figure 4f). The transplanted islet cells (separated from GFP-Tg) are shown in green, and the white dotted line indicates the range of the engrafted area. * The part represents the kidney cortex. Data were expressed as mean ± standard error (*: p <0.05, **: p <0.01).
Figures 5A-5C show whether angiogenesis at the transplanted site is due to donor or recipient origin. FIG. 5A is an outline of an experiment for evaluating the contribution of recipient blood vessels. For this experiment, GFP-Tg mice were used as recipients, and islets and early-EPCs were used as normal mice (donors) Respectively. In Figure 5B, CD31 antibody staining represents whole blood vessels at the transplantation site, and GFP antibody staining represents blood vessels from the recipient. The white dotted line shows the transplanted area, and the * part shows the kidney cortex. The bar bar size is 100 탆. Figure 5c shows the blood vessels formed quantitatively, the black bars represent the entire formed blood vessels and the white bars represent the blood vessels derived from the recipient with CD31 + / GFP + (**: p <0.01, †: p <0.05 vs . Islet cell alone).
Figures 6A-6D show the role of early-EPC transplanted with islet cells. 5A is an outline of an experiment for evaluating the contribution of the implanted early-EPC. Early-EPC derived from GFP-Tg was used to track the role of early-EPC, and normal mice were used for islet cells and recipient mice. FIG. 6B is a representative photograph showing that EPC derived from GFP-Tg grafted into CD31-stained blood vessels is embedded. The scale bar is 50 탆. The white dotted lines indicate the implanted site. FIG. 6c shows quantitatively early-EPC interfering with blood vessels, showing that it was not observed in islet-only transplantation group. Figure 6d shows that EPCs from GFP-Tg grafted with islet cells are present at the grafted site (left panel) and that the remaining EPCs express VEGF (middle panel). The scale bar is 100 탆.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Experimental animal

Green fluorescent protein-transgenic mice (GFP-Tg) derived from C57BL / 6J were purchased from Jackson Laboratory. Early -EPC and islet cells, shown in an overview, Figs. 5a and 6a for co-transplantation, the 10 to 12-week-old GFP-Tg mice and, depending on the experimental conditions, was used as donor and grantor, GFP-Tg mice Were used to assess the degree of contribution of blood vessels from the recipient. Mice were injected intraperitoneally with streptozotocin at 180 mg / kg to induce diabetes and mice maintained a sustained hyperglycemia (? 20 mmol / l) were used for transplantation. Blood glucose was measured by using a blood glucose meter. Animal management and experimental procedures were conducted in accordance with the provisions of the Animal Experiment Ethics Committee of Samsung Medical Center.

Isolation and culture of bone marrow-derived vascular endothelial progenitor cells (BM-EPC)

Mouse-derived bone marrow was obtained from the thighs and tibia. Muscles and connective tissues were cleanly removed from the bones and bone marrow in bone was obtained using a 30-gauge syringe. ACK lysis buffer was added to the obtained bone marrow to remove red blood cells. 2 (Endothelial Basal Medium-2) containing EGM-2 SingleQuots (EGM-2 medium, Lonza Inc, USA) and 5% FBS (Fetal Bovine Serum) were added to the obtained mononuclear cells. , And then inoculated in a 6-well plate coated with 2% gelatin at 1 × 10 ⁷ cells / well. After 3-4 days of incubation, unattached cells were washed with PBS and removed and replaced with fresh medium. BM-EPC cultured for 7-10 days confirmed the characteristics of endothelial progenitor cells by FACS and immunofluorescence staining. The identified BM-EPCs were transplanted with islet cells. GFP-Tg-derived EPC (GFP-EPC) and islet cells were simultaneously transplanted to assess the effect on angiogenesis.

Isolation of Islet Cells

Islet cells were isolated from GFP-Tg and normal mice. Islet cells were isolated by injecting 0.8 ㎎ / ㎏ of collagenase P (Roche, Germany) into the common bile duct, and the islet cells were purified using Ficoll density difference. The islet cells were cultured in M199 medium containing 10% FBS and 1% penicillin / streptomycin. One day later, islet cells were selected for transplantation.

Flow cytometry

Surface markers of mouse early-EPCs were analyzed by FACS (FACS Calibur). 5 × 10 ⁵ cells were reacted with 100 μl of the primary antibody or isotype control antibody diluted in 100 μl of 0.5% BSA for 20 min at 4 ° C and washed three times with PBS. The antibodies used were PE (phycoerythrin) -conjugated anti-mouse CD31, PE-anti-mouse CD117, PE-anti-mouse Flk-1 (VEGFR2) , FITC (fluorescence isothiocynate) -conjugated anti-mouse CD34 (eBioscience, USA), FITC-CD11b, and FITC-CD45 (BD Pharmingen). After washing and fixation, at least 10,000 cells were analyzed with CellQuestPro software.

RT-PCR analysis

Total RNA was extracted from the mononuclear cells and early-EPC separated from the bone marrow by TRIzol reagent (Invitrogen), cultured for 7 days, and then subjected to RT-PCR according to the manual of SuperScript II Reverse Transcriptase (Invitrogen, USA) with 1 μg of RNA. RT-PCR was performed using AccuPower PCR premix and each cDNA was amplified. The primer sequences used and the sizes of the PCR products were as follows:

CD31 (forward 5'-TGCAGGAGTCCTTCTCCACT-3 'and reverse 5'-ACGGTTTGATTCCACTTTGC-3', product size: 245 bp); Flk-1 (forward 5'-GGCGGTGGTGACAGTATCTT-3 'and reverse 5'-GTCACTGACAGAGGCGATGA-3', product size: 162 bp); vWF (forward 5'-CAGCATCTCTGTGGTCCTGA-3 'and reverse 5'-GATGTTGTTGTGGCAAGTGG-3', product size: 217 bp); eNOS (forward 5'-GACCCTCACCGCTACAACAT-3 'and reverse 5'-CTGGCCTTCTGCTCATTTTC-3', product size: 209 bp); And β-actin (forward 5'-TGTTACCAACTGGGACGACA-3 'and reverse 5'-GGGGTGTTGAAGGTCTCAAA-3', product size: 165 bp).

Immunochemical staining

Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and washed three times with PBS. The cells were blocked with PBS containing 5% normal serum and 0.1% Triton X-100 for 45 minutes. After blocking, primary antibody reaction was performed overnight at 4 ° C. The primary antibodies were rat anti-CD31 (1: 100, BD Phamingen), rat anti-VE-cadherin (CD144, 1: 100, BD Phamingen) and rabbit anti-Flk-1 (1: 100, Cell Signaling Technology). After washing three times with PBS, cells were incubated with Alexa-568-conjugated goat anti-rat and Alexa-568-conjugated goat anti-rabbit (1: 200, Molecular Probes) Secondary antibody reaction. Cell nuclei were visualized using DAPI (1: 2500, Molecular Probes).

Di I- ac - LDL Absorption capacity  And BS -1 lectin Cohesion

Early-EPCs were cultured in gelatin-coated 8 well chamber slides (Lab-Tek, Nunc, Germany). The adherent cells were incubated at 37 ° C for 1 hour with 10 μg / ml dil-ac-LDL (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein, Molecular Probes, USA) After incubation, the cells were fixed with 1% paraformaldehyde. Cells were washed 3 times with PBS and incubated with 10 ug / ml FITC (Fluorescein isothiocyanate) -conjugated mouse endothelial cell-specific BS-1 lectin (Bandeiraea simplicifolia lectin 1, Vector Laboratories, Lt; / RTI &gt; Fluorescence was visualized with a confocal microscope (Carl Zeiss Inc., USA) and a fluorescence microscope (Olympus, Japan).

Early- EPC  And Islet cell  Joint transplantation

Early-EPC and islet cells were collected in tubes (Eppendorf, USA) and centrifuged. Each suspension of early-EPC and pancreatic islets was pipetted and mixed, transferred to a PE (polyethylene) -50 tube using a Hamilton syringe and transplanted under the mouse kidney capsule. Diabetes-induced mice were conducting experiments in experimental outline in Fig. 4, 5 and 6 (Fig. 4-① 200 fluorescence islet cells, pancreatic cells, fluorescence ② 200 + 1 × 10 ⁶ normal early -EPC (the number of mice 2) normal islet cells + 1 × 10 ⁶ normal early-EPC (used as GFP-Tg recipient); FIG. 6-① 200 normal islet cells, 2 200 normal islets (using a normal mouse to be girls) cells + 1 × 10 ⁶ GFP- early -EPC). After transplantation, body weight and blood glucose were measured twice every two weeks.

After implantation in diabetic mice Islet cell  Functional inspection

Intraperitoneal glucose tolerance tests (IPGTT) were performed on days 14 and 28 after transplantation. After fasting for 10 hours, 1 g / kg glucose was injected into the peritoneal cavity and blood glucose was measured by time (0, 15, 30, 45, 60, 90 and 120 minutes). Blood was obtained at 0 min and 30 min via Retro-Orbital plexus, and the separated serum insulin was measured using an enzyme-linked immunosorbent assay (ELISA) kit. The percentage of mice that reached normal glucose levels and the time to reach (day) were calculated for each group and it was judged to be effective when the blood glucose level dropped below 11.1 mmol / l for 2 consecutive days during the blood glucose test. To demonstrate the transplantation dependence of whether diabetes in mice recovered from transplantation, renal resection with islet cell transplantation was performed.

Immunostaining and morphological analysis

After the transplantation, kidneys were removed at 14 and 28 days, fixed with 4% paraformaldehyde, washed with PBS, and immersed in 30% sucrose to prepare a frozen block. The tissue was cut into 10 ㎛ and the tissue was stained. Tissue staining was performed using an anti-insulin antibody for islet cell staining, an anti-CD31 antibody for new vascular staining, an anti-VEGF antibody as a growth factor involved in angiogenesis, an anti-GFP antibody for staining GFP- Anti-glucagon antibody was used for cell staining. The tissue was blocked with 10% normal goat serum and then incubated with rat anti-mouse CD31 antibody, rat polyclonal anti-GFP, rabbit anti-VEGF antibody, guinea pig anti-insulin antibody and rabbit anti- And used as a primary antibody. In addition, 568-conjugated goat anti-rat, 488 or 568-conjugated goat anti-rabbit antibody and Cy3-conjugated anti-guinea pig antibody were used as secondary antibodies. DAPI (4 ', 6-diamidino-2-phenylindole) was used for nuclear staining. Morphometric measurements and analysis were performed using Image-Pro Plus software version 5.1.

Statistical analysis

Data are expressed as mean ± standard error of measurement. The differences between the groups were analyzed by two-tailed unpaired Student's t- test and log-rank test. The p -value was <0.05 Were considered statistically significant.

Experiment result

Mouse Early - EPC  Investigate characteristics

Mouse early-EPCs were cultured in endothelial cell differentiation medium (EGM-2 BulletKit) for 7 days, and microscopic observation confirmed that the cells were spindle-shaped. Early-EPC confirmed the expression of endothelial cell marker proteins CD31, CD106 and Flk-1 as well as CD11b and CD45, which are markers of macrophages and mononuclear leukocytes, and CD34 and Sca-1, markers of blood stem cells, by FACS Respectively. In addition, these cells exhibited Di-ac-LDL uptake and BS-1 lectin binding ability, which are characteristic of EPC, and express the endothelial marker proteins CD31, Flk-1, vWF, eNOS and VE-cadherin by RT-PCR And immunofluorescence staining (Fig. 1 (a) to Fig. 1 (e)).

Diabetes mellitus cure rate diabetes reversal rate ) Improvement

We also investigated whether the number of pancreatic islet cells showing 50% diabetic cure rate after implantation was improved when co-transplanted with early-EPC. 100 islet cells did not show a blood glucose lowering effect (0/7) in the group transplanted alone, but 100% (3/3) of the blood glucose reduction effect was observed in the group transplanted with 300 transplanted islet cells. 200 islet cells alone showed a blood glucose lowering effect of 50% (6/12), and at the time of transplantation, 200 pancreatic islets were treated (Figs. 2a to 2c) . 200 out of 13 transplant recipients showed diabetic recovery, whereas 14 out of 17 transplanted group with early-EPC maintained normal glucose levels (Fig. 2d and 2f) . The mean blood glucose level of islet cells and EPCs was significantly lower (10.4 ± 0.7 mmol / L vs. 20.6 ± 2.9 mmol / L, p <0.001) than islet cell alone. As a result of insulin immunofluorescent staining in the pancreas extracted from the transplantation groups, almost no islet cells were found. This indicates that the remaining beta cells in the pancreas did not contribute to diabetic recovery due to regeneration, indicating that diabetic recovery was achieved by the transplanted islet cells. There was a significant difference in the time to reach normal blood glucose (17.6 ± 3.1 days versus 9.8 ± 1.4 days, p <0.05) between islet cell transplantation group and islet cell and early-EPC group. The cumulative percentage of normal glucose uptake over time was also higher in islet cell and early-EPC co-implanted group (Fig. 2g, P <0.05). After transplantation, body weight decreased in all groups, but the islet-cell and early-EPC transplantation groups were significantly increased over time compared to the islet-only transplantation group. EPC alone and non - grafted diabetic group failed to maintain blood glucose levels and were eventually euthanized by excessive weight loss. EPC (non-endothelial progenitor cells) from bone marrow were also examined for the ability to regulate blood glucose levels when they were co-transplanted with islet cells. As a result, islet cell and non-EPC co- (Fig. 2E).

Islet cell Than single transplant  Enhancements

Intraperitoneal glucose tolerance tests (IPGTT) were performed to investigate the function of transplanted islet cells in vivo. On the 14th and 28th day after transplantation, the islet cell and early-EPC co-transplantation group showed an improved blood glucose change after glucose loading compared with the islet cell transplantation group, and there was a significant difference at all time intervals 3a). In addition, the value of the area of the glucose curve (AUG _glu ) in the glucose tolerance test was significantly lower in islet cell and early-EPC transplantation group than islet transplantation group ( p <0.01 , Fig. 3b). We examined whether the improved glycemic control of islet cells and early-EPC transplantation was due to increased insulin production from transplanted islet cells. In islet cell and early-EPC transplantation group, serum insulin concentration in fasting serum was significantly higher than islet transplantation group (0 min, p <0.05; 30 min, p <0.01). These results show that the islet cell and the early-EPC joint transplantation can provide enhanced function than the islet cell alone group (Fig. 3c).

Islet  Normal shape preservation and angiogenesis

To assess the degree of angiogenesis, the kidneys were harvested at day 28. First, the endocrine and non-endocrine regions were evaluated. Since the transplanted GFP-islet cells corresponded to the insulin staining portion, the GFP portion was evaluated as the endocrine region. Islet cells and early-EPCs were significantly larger in the endocrine and non-endocrine regions than in the islet cells alone, and the shapes of the endocrine regions were also different. The co-transplant group was divided into several lumps, and the islet-only transplant group was found to be a loose lump ( p < 0.05) (Figs. 4A and 4B). In addition, glucagon-positive alpha cells were mostly distributed around the endocrine region in the co-transplantation group, similar to the location of alpha cells in pancreatic islet cells (Fig. 4c). On the other hand, alpha - cells were irregularly distributed in the islet transplantation group. There was a significant difference in the number of alpha cells between the two groups ( p <0.01) (Fig. 4d). To compare angiogenic density, tissues used CD31 antibody. Vessel density was significantly higher in the co-transplant group than in the islet transplant alone group, and blood vessels were observed in the endocrine area and in the periphery (p <0.01) (Figs. 4E and 4F). In addition, AUG _glu induced by glucose _tolerance test showed correlation with insulin-positive area and vessel density. These data suggest that pancreatic islet cells and early-EPC joint transplantation can improve the preservation of the shape of established endocrine cells and post-transplantation angiogenesis and islet cell fixation.

New vessel Formative  Improving

Joint transplantation of islet cells and early-EPCs showed improved angiogenesis from established islet cells. However, we did not know how much blood vessels from the recipient contributed. Therefore, GFP-Tg mice were used as recipients for easy identification of recipient-derived blood vessels. After transplantation, tissues were stained with anti-CD31 antibody and anti-GFP antibody on days 14 and 28. In the staining for CD31 (staining of vessels throughout the fixed area), vascular density was significantly higher in islet cell and early-EPC co-implanted group than in islet cell alone group ( p <0.01) The density of blood vessels (CD31 + / GFP +) also increased in the pancreatic cell + EPC group ( p <0.05) (Fig. 5b and Fig. 5c). However, donor-derived vascular density accounted for less than 40% of all CD31-positive vessels. These results indicate that angiogenesis is predominantly involved in donor - derived angiogenesis, rather than recipient - derived blood vessels.

Promotion of neovascularization

EPCs were continuously present in the transplanted area and, if present, their function. For this experiment, the presence of GFP-Tg-derived early-EPCs in the transplanted area was readily ascertained. The transplanted GFP-early-EPC was found to be present in or around the endocrine region and EPCs in the blood vessels were observed (4.2 +/- 0.79 GFP + / CD31 +) (Figures 6b and 6c). Sometimes GFP-early-EPC was also observed in the kidney cortex. The transplanted early-EPCs did not undergo insulin staining, indicating that they did not differentiate into insulin-secreting cells. EPC remaining after transplantation showed expression of VEGF (Fig. 6d). These results suggest that co-implanted early-EPC can enhance angiogenesis at the transplanted site by both the paracrine effect and the direct mechanism of angiogenic incorporation.

references

1. Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343: 230-238.

2. Ryan EA, Paty BW, Senior PA et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54: 2060-2069.

3. Jansson L, Carlsson PO. Graft vascular function after transplantation of pancreatic islets. Diabetologia 2002; 45: 749-763.

4. Vajkoczy P, Olofsson AM, Lehr HA et al. Histogenesis and ultrastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial cells of host origin. Am J Pathol 1995; 146: 1397-1405.

5. Takahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999; 5: 434-438.

6. Kawamoto A, Gwon HC, Iwaguro H et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001; 103: 634-637.

7. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003; 9: 702-712.

8. Hur J, Yoon CH, Kim HS et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 2004; 24: 288-293.

9. Yoder MC, Mead LE, Prater D et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem / progenitor cell principals. Blood 2007; 109: 1801-1809.

10. Ingramda, Caplice NM, Yoder MC. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood 2005; 106: 1525-1531.

11. Sekiguchi H, Ii M, Losordo DW.The relative potency and safety of endothelial progenitor cells and unselected mononuclear cells for recovery from myocardial infarction and ischemia. J Cell Physiol 2009 219: 235-242.

12. Scolding N, Marks D, Rice C. Estrogenic mesenchymal bone marrow stem cells: practical consideration. J Neurol Sci 2008 2265: 111-115.

13. Siatskas C, Payne NL, Short MA, Bernard CC. A consensus statement addressing mesenchymal stem cell transplantation for multiple sclerosis: it's time !. Stem Cell Rev 2006 6: 500-506.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (15)

A method for producing a plant cell for an islet cell transplantation comprising the steps of:
(a) culturing mammalian early-endothelial progenitor cells;
(b) culturing mammalian islet cells (Islet Cells); And
(c) mixing the cultured pre-endothelial progenitor cells and the islet cells to prepare a cell plant.
2. The method of claim 1, wherein the mammal is a human, a bovine, a horse, a pig, a goat, a dog, a cat, a chicken, a mouse, a rat, a rabbit or a guinea pig.
2. The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) are derived from bone marrow.
2. The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) are spindle-shaped.
3. The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) express CD106, CD31, Flk-1, eNOS, vWF and VE-cadherin.
2. The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) express CD11b, CD45, CD34 and Sca-1.
2. The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) have DiI-ac-LDL absorption capacity and BS-1 lectin binding capacity.
The method of claim 1, wherein the pre-endothelial progenitor cells of step (a) are cultured for 5-12 days.
2. The method of claim 1, wherein the islet cells of step (b) are cultured for 16-24 hours.
2. The method according to claim 1, wherein the cell plant of step (c) is a cell population of early-endothelial progenitor cells and an islet cell mixture of 1000: 1 to 10000: 1.
2. The method of claim 1, wherein the mixing of step (c) is performed immediately prior to implantation.
2. The method according to claim 1, wherein the plant cell has a normal glucose uptake capacity of 1.5 to 3.0 times as much as an islet cell alone.
2. The method according to claim 1, wherein the plant cell has an angiogenesis ability of 2 to 3 times as much as an islet cell of an islet cell.
A therapeutic cell plant for the transplantation of an islet cell produced by the method of any one of claims 1 to 13.
13. A pharmaceutical composition for improving, preventing or treating diabetes comprising a cell plant produced by the method of any one of claims 1 to 13.

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